C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro.

With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.

Dopamine and adenosine 3',5'-monophosphate responses of single mammalian sympathetic neurons.

Acetylcholine (ACh), dopamine, and dibutyryl-adenosine 3',5'-monophosphate (dbcAMP) were applied iontophoretically to the rabbit superior cervical ganglion cells from triple-barreled micropipettes, and the response was recorded by intracellular techniques. All ganglion cells tested responded to the depolarizing action of ACh, whereas less than half of the cells that responded to ACh were hyperpolarized by dopamine. This effect was blocked by low concentrations of haloperidol. None of the cells examined responded to dbcAMP applied by iontophoresis. Hence, the present result is not consistent with the concept that a cyclic AMP mechanism underlies the hyperpolarizing effect of dopamine.

Substance P: a putative sensory transmitter in mammalian autonomic ganglia.

Repetitive presynaptic stimulation elicited slow membrane depolarization in neurons of inferior mesenteric ganglia from guinea pigs. This response was not blocked by cholinergic antagonists but was specifically and reversibly inhibited by a substance P analog, (D-Pro2, D-Phe7, D-Trp9)-substance P, which also depressed the depolarization induced by exogenously applied substance P. The atropine-sensitive slow excitatory and slow inhibitory postsynaptic potentials evoked in neurons of rabbit superior cervical ganglia were not affected by the substance P analog. These and previous results provide strong support for the hypothesis that substance P or a closely related peptide is the transmitter mediating the slow depolarization. The latter may represent a sensory input from the gastrointestinal tract to neurons of the prevertebral ganglia.

Excitatory effects of human immunodeficiency virus 1 Tat on cultured rat cerebral cortical neurons.

Human immunodeficiency virus 1 (HIV-1) Tat protein is one of the neurotoxins involved in the pathogenesis of HIV-1-associated neuronal disorders. Combined electrophysiological and optical imaging experiments were undertaken to investigate whether HIV-1 Tat30-86, herein referred to as Tat30-86, acted directly or indirectly via the release of glutamate or both and to test its effect on the properties of spontaneous quantal events in cultured cortical neurons. Whole-cell patch recordings were made from cultured rat cortical neurons in either current- or voltage-clamp mode. Tat30-86 (50-1000 nM) induced in a population of cortical neurons a long-lasting depolarization, which was accompanied by a decrease of membrane resistance and persisted in a Krebs solution containing tetrodotoxin (TTX, 0.5 microM). Depolarizations were slightly reduced by pretreatment with glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM) and d-2-amino-5-phosphonovaleric acid (AP-5) (50 microM), and were markedly reduced in a Ca(2+)-free Krebs solution; the differences were statistically significant. Tat30-86-induced inward currents had a reversal potential between -30 and 0 mV. While not causing a noticeable depolarization, lower concentrations of Tat30-86 (10 nM) increased membrane excitability, as indicated by increased numbers of neuronal discharge in response to a step depolarizing pulse. Tat30-86 (10 nM) increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), while not significantly affecting their amplitude. Tat30-86 (10 nM) moderately increased the frequency as well as the amplitude of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Ratiometric Ca(2+) imaging studies showed that Tat30-86 produced three types of Ca(2+) responses: 1) a fast and transitory increase, 2) Ca(2+) oscillations, and 3) a fast increase followed by a plateau; the glutamate receptor antagonists eliminated the late component of Ca(2+) response. The result suggests that Tat30-86 is an active fragment and that it excites cortical neurons directly and indirectly via releasing glutamate from adjacent neurons.

Distribution and ultrastructural localization of GEC1 in the rat CNS.

We have previously demonstrated that GEC1 interacts with the kappa opioid receptor and GEC1 expression enhances cell surface expression of the receptor in Chinese hamster ovary cells. In this study, we generated an antiserum (PA629) directed against GEC1 in rabbits, characterized its specificity, and investigated distribution of GEC1 in tissues and in brain regions and spinal cord and its subcellular localization in hypothalamic neurons in the rat. Immunofluorescence staining demonstrated that PA629 recognized HA-GEC1 transfected into Chinese hamster ovary cells, but not HA-GABARAP or HA-GATE-16, although the three share high homology. Pre-incubation of PA629 with GST-GEC1, but not GST, abolished the staining. In immunoblotting, affinity-purified PA629 (PA629p) recognized GEC1, GABARAP and GATE-16. GEC1 migrated slower than GABARAP and GATE-16, with a M(r) of 16 kDa for GEC1 and M(r) of 14 kDa for GABARAP and GATE-16. Immunoblotting results showed that GEC1 level was higher in liver and brain than in lung and heart, and very low in kidney and skeletal muscle. GEC1 was present in all rat brain regions examined and spinal cord. Immunohistochemistry demonstrated that GEC1 immunoreactivity was distributed ubiquitously in the rat CNS with highly intense immunoreactivity in various brain nuclei and motor neurons of the spinal cord. Ultrastructural examination of neurons in the paraventricular nucleus of the hypothalamus showed that GEC1 was associated with endoplasmic reticulum and Golgi apparatus and distributed along plasma membranes and in cytosol. Coupled with our previous observation that GEC1 interacts with N-ethylmaleimide-sensitive factor, these findings strongly suggest that GEC1 functions in intracellular trafficking in the biosynthesis pathway and perhaps also the endocytic pathway. The widespread distribution of GEC1 suggests that GEC1 may be associated with many proteins, in addition to the kappa opioid receptor.

Phoenixin: A candidate pruritogen in the mouse.

Phoenixin (PNX) is a 14-amino acid amidated peptide (PNX-14) or an N-terminal extended 20-residue amidated peptide (PNX-20) recently identified in neural and non-neural tissue. Mass spectrometry analysis identified a major peak corresponding to PNX-14, with negligible PNX-20, in mouse spinal cord extracts. Using a previously characterized antiserum that recognized both PNX-14 and PNX-20, PNX-immunoreactivity (irPNX) was detected in a population of dorsal root ganglion (DRG) cells and in cell processes densely distributed to the superficial layers of the dorsal horn; irPNX cell processes were also detected in the skin. The retrograde tracer, Fluorogold, injected subcutaneously (s.c.) to the back of the cervical and thoracic spinal cord of mice, labeled a population of DRG, some of which were also irPNX. PNX-14 (2, 4 and 8 mg/kg) injected s.c.to the nape of the neck provoked dose-dependent repetitive scratching bouts directed to the back of the neck with the hindpaws. The number of scratching bouts varied from 16 to 95 in 30 min, commencing within 5 min post-injection and lasted 10-15 min. Pretreatment of mice at -20 min with nalfurafine (20 µg/kg, s.c.), the kappa opioid receptor agonist, significantly reduced the number of bouts induced by PNX-14 (4 mg/kg) compared with that of saline-pretreated mice. Our results suggest that the peptide, PNX-14, serves as one of the endogenous signal molecules transducing itch sensation in the mouse.

Facilitation and inhibition of nicotinic transmission by eserine in the sympathetic ganglia of the rabbit.

The effects of eserine on neurons and on ganglionic transmission of the isolated superior cervical ganglia of the rabbit were investigated by means of intracellular recording techniques. At the concentration of 10 microM or less, eserine reversibly increased the amplitude and duration of the fast excitatory postsynaptic potential (f-epsp) induced by preganglionic nerve stimulation and of the membrane depolarization evoked by iontophoretically-applied acetylcholine (ACh), but not carbachol. At the concentration of 50 microM or more, eserine consistently and reversibly depressed the fast excitatory postsynaptic potential as well as the depolarization induced by iontophoretic application of either ACh or carbachol. Furthermore, depolarization by ACh evoked in a low Ca/high Mg solution, which blocked the liberation of transmitter was similarly reduced by eserine in greater concentrations. The passive membrane properties of the sympathetic neurons were not significantly altered by eserine in the majority of neurons studied. The results indicate that the facilitatory action of eserine on ganglionic transmission may be explained by its anticholinesterase activity, whereas eserine-induced block of transmission appears to be related to a direct interaction between the compound and the postsynaptic ACh receptor-channel complex.

Post- and presynaptic GABA(B) receptor activation in neonatal rat rostral ventrolateral medulla neurons in vitro.

Whole-cell patch recordings were made from immature (six- to 12-day-old) rat rostral ventrolateral medulla neurons in brainstem slices. GABA or the specific GABA(B) receptor agonist (-)baclofen (10-50 microM) by superfusion or by pressure ejection induced an outward current or a hyperpolarization, which persisted in a tetrodotoxin (0.3 microM)-containing Krebs' solution in nearly every cell tested. The GABA(B) receptor antagonists 2-hydroxy saclofen (50-200 microM) and CGP 35348 (50-200 microM) dose-dependently suppressed baclofen-currents. Baclofen-currents were suppressed by barium (1 mM) but not by tetraethylammonium (20 mM), low Ca2+ (0.24 mM) solution or in a solution containing the Ca2+ chelator BAPTA-AM (10 microM). The outward current had an estimated reversal potential of -98, -77 and -52 mV in 3.1, 7 and 15 mM [K+]o. Pre-incubation of slices with pertussis toxin (500 microg/ml for 5-7 h) or intracellular dialysis with GDP-beta-S (500 microM) markedly reduced baclofen-currents. Baclofen in low concentrations (1-3 microM) that caused slight or no change of holding currents and of inward or outward currents induced by exogenously applied glutamate or glycine/GABA, decreased excitatory and inhibitory postsynaptic currents by an average of 86.5 +/- 4.3% and 78.4 +/- 2.7%. The GABA(B) antagonist CGP 35348 (100 microM) increased the excitatory postsynaptic currents by an average of 64%, without causing a significant change in holding currents in 10/18 cells tested. Our results indicate the presence of post- and presynaptic GABA(B) receptors in the rostral ventrolateral medulla neurons. Activation of postsynaptic GABA(B) receptors induces an outward K+ current which is barium-sensitive, Ca2+-independent and may be coupled to a pertussis-sensitive G-protein. Activation of presynaptic GABA(B) receptors attenuates excitatory or inhibitory synaptic transmission. More importantly, the observation that CGP 35348 enhanced excitatory synaptic currents implies a removal of tonic activation of presynaptic GABA(B) receptors by endogenously released GABA (disinhibition), supporting the hypothesis that these receptors may have a physiological role in regulating the input and output ratio in a subset of rostral ventrolateral medulla neurons in vivo.

Serotonin modulates synaptic transmission in immature rat ventrolateral medulla neurons in vitro.

Patch-clamp recordings in whole-cell configuration were made from ventrolateral medulla neurons of brainstem slices from 8-12-day-old rats. 5-Hydroxytryptamine (3-30 microM) concentration-dependently suppressed excitatory and inhibitory postsynaptic currents evoked by focal stimulation. An augmentation of inhibitory synaptic currents by 5-hydroxytryptamine was noted in a small number of neurons. 5-Hydroxytryptamine depressed synaptic currents with or without causing a significant change in holding currents and membrane conductances; the inward or outward currents induced by exogenously applied glutamate or GABA/glycine were also not significantly changed by 5-hydroxytryptamine. In paired-pulse paradigms designed to evaluate a presynaptic site of action, 5-hydroxytryptamine suppressed synaptic currents but enhanced the paired-pulse facilitation. 5-Hydroxytryptamine reduced the frequency of miniature excitatory postsynaptic currents without significantly affecting the amplitude. 5-Carboxamidotryptamine, 8-hydroxy-2(di-n-propylamino)tetralin, sumatriptan and N-(3-trifluoromethylphenyl)piperazine which exhibit 5-hydroxytryptamine1 receptor agonist activity, depressed synaptic currents with different potencies, with 5-carboxamidotryptamine being the most potent. The non-selective 5-hydroxytryptamine1 receptor antagonist pindolol attenuated the presynaptic effect of 5-hydroxytryptamine, whereas the 5-hydroxytryptamine1A antagonist pindobind-5-hydroxytryptamine1A and 5-hydroxytryptamine2 receptor antagonist ketanserin were ineffective. Our results indicate that 5-hydroxytryptamine suppressed synaptic transmission in ventrolateral medulla neurons by activating presynaptic 5-hydroxytryptamine1 receptors, probably the 5-hydroxytryptamine1B/5-hydroxytryptamine1D subtype. In addition, 5-hydroxytryptamine augmented inhibitory synaptic currents in a small number of neurons the site and mechanism of this potentiating action are not known.

Nitric oxide synthase immunoreactivity in rat pontine medullary neurons.

Nitric oxide synthase immunoreactivity was detected in neurons and fibers of the rat pontine medulla. In the medulla, nitric oxide synthase-positive neurons and processes were observed in the gracile nucleus, spinal trigeminal nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, nucleus ambiguus, medial longitudinal fasciculus, reticular nuclei and lateral to the pyramidal tract. In the pons, intensely labeled neurons were observed in the pedunculopontine tegmental nucleus, paralemniscal nucleus, ventral tegmental nucleus, laterodorsal tegmental nucleus, and lateral and medial parabrachial nuclei. Labeled neurons and fibers were seen in the interpeduncular nuclei, dorsal and median raphe nuclei, central gray and dorsal central gray, and superior and inferior colliculi. Double-labeling techniques showed that a small population (< 5%) of nitric oxide synthase-positive neurons in the medulla also contained immunoreactivity to the aminergic neuron marker tyrosine hydroxylase. The majority of nitric oxide synthase-immunoreactive neurons in the dorsal and median raphe nuclei were 5-hydroxytryptamine-positive, whereas very few 5-hydroxytryptamine-positive cells in the caudal raphe nuclei were nitric oxide synthase-positive. Virtually all nitric oxide synthase-positive neurons in the pedunculopontine and laterodorsal tegmental nuclei were also choline acetyltransferase-positive, whereas nitric oxide synthase immunoreactivity was either low or not detected in choline acetyltransferase-positive neurons in the medulla. The results indicate a rostrocaudal gradient in the intensity of nitric oxide synthase immunoreactivity, i.e. it is highest in neurons of the tegmentum nuclei and neurons in the medulla are less intensely labeled. The majority of cholinergic and serotonergic neurons in the pons are nitric oxide synthase-positive, whereas the immunoreactivity was either too low to be detected or absent in the large majority of serotonergic, aminergic and cholinergic neurons in the medulla.

Nitric oxide synthase-immunoreactive vagal afferent fibers in rat superior cervical ganglia.

Chronic (5-14 days) preganglionic denervation of the rat superior cervical ganglia by sectioning the cervical sympathetic trunk resulted in a time-related partial or complete loss of nitric oxide synthase (isoform I)-immunoreactive fibers and terminals surrounding many sympathetic ganglionic neurons. Unexpectedly, denervation unmasked many varicose nitric oxide synthase-immunoreactive fibers, some of which could be traced the entire length of the superior cervical ganglia. Injection of the retrograde tracer Fluorogold into the superior cervical ganglia labeled a population of nodose ganglion cells and of dorsal root ganglion cells from C8 to T3 segments. When the same sections were processed for nitric oxide synthase-immunoreactivity, 40% of the Fluorogold-containing nodose ganglion cells also expressed nitric oxide synthase-immunoreactivity, whereas colocalization was observed in only a few dorsal root ganglion cells. Similarly, injection of Fluorogold into denervated superior cervical ganglia labeled a population of nodose ganglion cells. Sectioning of all nerve trunks associated with the superior cervical ganglion prior to injection of Fluorogold, except the cervical sympathetic trunk, resulted in no detectable labeling of Fluorogold in the ipsilateral nodose ganglion cells. These results indicate that a population of rat nodose ganglion cells contain nitric oxide synthase and that some of these neurons project their axons through the superior cervical ganglion and terminate in the peripheral target tissues. The possibility that nitric oxide synthase-immunoreactive vagal afferent fibers may participate in nociception is considered.

Nitric oxide and excitatory postsynaptic currents in immature rat sympathetic preganglionic neurons in vitro.

Neuronal nitric oxide synthase immunoreactivity was localized to sympathetic preganglionic neurons of the intermediolateral cell column and cyclic GMP immunoreactivity to nerve fibers projecting into the intermediolateral cell column of 20-25-day-old rats. Whole-cell patch-clamp recordings were made from sympathetic preganglionic neurons in spinal cord slices of immature rats and the role of nitric oxide and cyclic GMP on excitatory postsynaptic currents was studied. Superfusing the slices with the nitric oxide precursor L-arginine (300 microM) increased the amplitude of evoked excitatory postsynaptic currents as well as the frequency of spontaneous miniature excitatory postsynaptic currents in some neurons from minutes to over 1 h. The nitric oxide synthase inhibitor N(W)-nitro-L-arginine (100 microM) and the nitric oxide scavenger hemoglobin (100 microM) antagonized the potentiating effect of L-arginine. The nitric oxide donor sodium nitroprusside (100 microM) potentiated the synaptic currents in a manner similar to that of L-arginine and this effect was blocked by hemoglobin. The membrane-permeable cyclic GMP analogue dibutyryl guanosine 3',5'-cyclic monophosphate (350 microM), in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (750 microM), potentiated the evoked excitatory postsynaptic currents and increased the frequency of miniature excitatory postsynaptic currents; these effects were not prevented by hemoglobin. The results indicate that nitric oxide may facilitate the release of excitatory transmitters, possibly through a presynaptic cyclic GMP-dependent mechanism.

Pituitary adenylate cyclase activating polypeptide immunoreactivity in the rat spinal cord and medulla: implication of sensory and autonomic functions.

Immunoreactivity to pituitary adenylate cyclase activating polypeptide-38 was detected in numerous nerve fibres in layers I and II of the dorsal horn of the rat and some of these fibres extended into the deeper layers of all segments of the spinal cord. Immunoreactivity was also detected in the lateral funiculus projecting into the intermediolateral cell column of the lower cervical and thoracic segments and in the lateral pathway terminating in the intermediate gray area of the lower lumbar and sacral segments. Neurons in the lateral horn area were not immunoreactive nor were the ventral horn motoneurons. In the medulla, numerous immunoreactive fibres were observed in the spinal trigeminal tract and superficial layers of the caudal spinal trigeminal nucleus but few in the interpolar spinal trigeminal nucleus. A prominent immunoreactive nerve bundle emanated from the caudal spinal trigeminal nucleus and projected into the solitary tract. A dense network of immunoreactive neurons and fibres was present in the nucleus raphe obscurus, lateral reticular nucleus and parvocellular lateral reticular nucleus. Immunoreactive fibres could also be detected in the solitary tract and area postrema. Labelled somata were occasionally noted in various subnuclei of the nucleus of the solitary tract and nucleus raphe pallidus. In addition, a small number of positive neurons were detected in an area between the lateral reticular nucleus and inferior olive and near the ventral surface of the medulla (parapyramidal region). A few weakly-labelled cells were occasionally seen in the dorsal motor nucleus of vagus. A population of neurons in the trigeminal, nodose and dorsal root ganglia from all segments of the spinal cord displayed low to intense immunoreactivity. The presence of immunoreactivity in nodose and dorsal root ganglia, dorsal horn, spinal autonomic nuclei, solitary tract and in certain areas of the medulla suggests that this peptide may participate in a variety of sensory and autonomic functions.

Neurokinin A in capsaicin-sensitive neurons of the guinea-pig inferior mesenteric ganglia: an additional putative mediator for the non-cholinergic excitatory postsynaptic potential.

The presence of neurokinin-A-like immunoreactivity in guinea-pig inferior mesenteric ganglia was detected by radioimmunoassay procedures. Pretreating the animals with capsaicin 7 days prior to experimentations reduced the mean content of neurokinin-A-like immunoreactivity by 85% from its control value of 150 +/- 31.3 fmol per ganglion. High-performance liquid chromatography revealed that neurokinin-A-like immunoreactivity was heterogenous as in addition to neurokinin A, peaks corresponding to the amphibian tachykinin eledoisin and to neuropeptide K were detected, and they too were depleted by capsaicin. Electrophysiological studies showed that neurokinin A applied either by superfusion or by pressure ejection evoked a slow depolarization in the majority of inferior mesenteric ganglia neurons in vitro. Neurokinin-A-evoked depolarizations in the majority of cells tested were associated with a small increase in membrane input resistance. However, the responses were increased by membrane hyperpolarization: the extrapolated mean equilibrium potential of neurokinin-A-induced depolarization was -36 mV. Removal of extracellular sodium but not chloride ions suppressed the neurokinin-A-induced depolarization. The slow depolarization elicited either by exogenously applied substance P or by repetitive stimulation of hypogastric nerves was reversibly eliminated in the presence of neurokinin A. Collectively, our studies suggest that neurokinin-A-like immunoreactivity may coexist with substance-P-like immunoreactivity in capsaicin-sensitive fibers in the guinea-pig prevertebral ganglia and that the similarity of the actions of neurokinin A on the one hand and substance P on the other raises the possibility that non-cholinergic excitatory potentials elicited in the inferior mesenteric ganglia may be generated by not one but a number of closely related tachykinins.

Nociceptin-like immunoreactivity in the rat dorsal horn and inhibition of substantia gelatinosa neurons.

Nociceptin, also referred to as orphanin FQ, is believed to be the endogenous ligand for the ORL1. Nociceptin, when injected intracerebroventricularly to mice, produced hyperalgesia in behavioral tests. Recent studies have demonstrated the presence of ORL1 transcript in the spinal cord, and ORL1-like immunoreactivity has been localized to nerve fibers and somata throughout the spinal cord. Here, we report the localization of nociceptin-like immunoreactivity to fiber-like elements of the superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell recordings from substantia gelatinosa neurons in transverse lumbar spinal cord slices of 22-26-day-old rats showed that exogenous nociceptin at low concentrations (100-300 nM) depressed excitatory postsynaptic potentials evoked by stimulation of dorsal rootlets without causing an appreciable change of resting membrane potentials and glutamate-evoked depolarizations. At a concentration of 1 microM, nociceptin hyperpolarized substantia gelatinosa neurons and suppressed spike discharges. The hyperpolarizing and synaptic depressant action of nociceptin was not reversed by the known opioid receptor antagonist naloxone (1 microM). Our result provides evidence that nociceptin-like peptide is concentrated in nerve fibers of the rat dorsal horn and that it may serve as an inhibitory transmitter within the substantia gelatinosa.

GABA- and glycine-mediated inhibitory postsynaptic potentials in neonatal rat rostral ventrolateral medulla neurons in vitro.

Whole-cell patch recordings were made from rostral ventrolateral medulla neurons of two in vitro preparations: (i) brainstem spinal cords of two- to five-day-old rats, and (ii) coronal brainstem slices of eight- to 12-day-old rats, and the inhibitory synaptic activities in these neurons have been studied. In brainstem spinal cord preparations, Lucifer Yellow was diffused into the recording neurons at the end of experiments. Medullary neurons were characterized as: (i) spinally projecting by the appearance of an antidromic spike following electrical stimulation of the spinal tract between T2 and T3 segments, and (ii) adrenergic by the detection of phenylethanolamine-N-methyltransferase immunoreactivity in Lucifer Yellow-filled neurons. Of the 13 spinally projecting and phenylethanolamine-N-methyltransferase-positive medullary neurons, focal stimulation elicited in the presence of glutamate receptor antagonists an inhibitory postsynaptic potential in nine neurons. Inhibitory synaptic potentials were reversibly eliminated by the GABA(A) receptor antagonist bicuculline (10-20 microM) in six of nine neurons, by the glycine receptor antagonist strychnine (0.1-1 microM) in two and by a combination of bicuculline and strychnine in one neuron. In brainstem slice preparations, focal stimulation elicited three types of synaptic potential: (i) an excitatory postsynaptic potential, (ii) an inhibitory postsynaptic potential and (iii) a biphasic synaptic potential consisting of an excitatory synaptic potential followed by an inhibitory synaptic potential. Inhibitory synaptic potentials had a reversal potential between -70 and -80 mV, reversed their polarity in a low (6.7 mM) Cl- Krebs' solution, and suppressed or blocked by either bicuculline or strychnine or both. Elimination of inhibitory synaptic potentials unmasked in some cells an excitatory synaptic potential or enhanced the excitatory synaptic potential component in medullary neurons with a biphasic response, indicating a marked convergence of excitatory and inhibitory inputs onto a single neuron. A population of medullary neurons appeared to be pacemaker neurons whereby they discharged spontaneously. When discharges were suppressed by membrane hyperpolarization, focal stimulation elicited inhibitory synaptic potentials in 8/23 neurons tested. Our results suggest that inhibitory synaptic potentials in medullary neurons are mediated by either GABA and/or glycine which open primarily Cl- channels. The prevalence of inhibitory synaptic potentials in medullary neurons indicates an essential role of inhibitory transmission in controlling the input and output ratio of these neurons.

Endomorphin-like immunoreactivity in the rat dorsal horn and inhibition of substantia gelatinosa neurons in vitro.

Endomorphin 1 and 2 are two tetrapeptides recently isolated from bovine as well as human brains and proposed to be the endogenous ligand for the mu-opiate receptor. Opioid compounds expressing mu-receptor preference are generally potent analgesics. The spinal cord dorsal horn is considered to be an important site for the processing of sensory information including pain. The discovery that endomorphins produced greater analgesia in mice upon intrathecal as compared to intracerebroventricular injections raises the possibility that dorsal horn neurons may represent the anatomic site upon which endomorphins exert their analgesic effects. We report here the detection of endomorphin 2-immunuoreactive fiber-like elements in superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell patch recordings from substantia gelatinosa neurons of cervical spinal cord slices revealed two conspicuous effects of exogenously applied endomorphin 1 and 2: (i) depression of excitatory postsynaptic potentials evoked by stimulation of dorsal root entry zone, and (ii) hyperpolarization of substantia gelatinosa neurons. These effects were reversed by the selective mu-opiate receptor antagonist beta-funaltrexamine. Collectively, the detection of endomorphin-like immunoreactivity in nerve fibers of the superficial layers and the inhibitory action of endomorphins on substantia gelatinosa neurons provide further support for a potential role of these two peptides in spinal nociception.

Nitric oxide synthase immunoreactivity in the rat, mouse, cat and squirrel monkey spinal cord.

The distribution of nitric oxide synthase-immunoreactive neurons was examined in the spinal cord of rats, mice, cats and squirrel monkeys at the light microscopic level. Some sections were processed for choline acetyltransferase immunoreactivity. Double-labeling techniques were used to assess possible co-localization of nitric oxide synthase and choline acetyltransferase immunoreactivity in the same spinal neurons. Nitric oxide synthase-immunoreactive neurons were concentrated in three fairly well-defined regions of the spinal cord of all species studied: (i) the intermediolateral cell column of the thoracic and sacral segments, (ii) lamina X of all segments, and (iii) the superficial layers of the dorsal horn of all segments. A few nitric oxide synthase-immunoreactive neurons were scattered in the deeper laminae and the ventral horn. There were fewer nitric oxide synthase-positive neurons in monkey spinal lamina X and dorsal horn than in similar locations of rodents and felines. Double-staining showed that not all choline acetyltransferase-positive neurons in the intermediate cell column and lamina X were nitric oxide synthase-immunoreactive. In the ventral horn, choline acetyltransferase-positive neurons (presumed motoneurons) were nitric oxide synthase-negative. In addition to cell bodies, nitric oxide synthase-positive fibers were scattered in the dorsal, lateral and ventral horns of all species. Finally, punctate nitric oxide synthase-immunoreactive fibers were seen traversing the dorsal, lateral and ventral white matter, and reaching the respective gray matter. The present study shows that, in spite of quantitative differences, the pattern of distribution of nitric oxide synthase-positive neurons in the spinal cord was similar across the four species. The concentration of nitric oxide synthase-positive neurons in the autonomic nuclei and laminae I, II and X of all four species underscores a prominent role of these neurons in visceral and sensory functions.

Phaclofen-insensitive presynaptic inhibitory action of (+/-)-baclofen in neonatal rat motoneurones in vitro.

1. Intracellular recordings were made from antidromically identified motoneurones in transverse spinal cord slices from neonatal (12-16 day) rats. 2. Superfusion of (+/-)-baclofen (0.5-50 microM) reduced the excitatory postsynaptic potentials (e.p.s.ps) and inhibitory postsynaptic potentials (i.p.s.ps) evoked by dorsal root or dorsal root entry zone stimulation in a concentration-dependent manner; the calculated EC50 was 2.4 microM. Baclofen in comparable concentrations also reversibly eliminated spontaneously occurring e.p.s.ps and i.p.s.ps. 3. (-)-Baclofen was more effective as compared to baclofen in reducing the synaptic responses, whereas (+)-baclofen at concentrations as high as 50 microM was ineffective. 4. Baclofen (less than 5 microM) attenuated the synaptic responses without causing a significant change of passive membrane properties and depolarizations induced by exogenously applied glutamate. In addition to synaptic depression, baclofen (greater than 5 microM) caused a hyperpolarization associated with decreased membrane resistance in some of the motoneurones; the glutamate responses were also attenuated. 5. Baclofen reversibly depressed the spike after-hyperpolarization of the motoneurones. 6. GABA (1-10 mM) depressed synaptic transmission and depolarized or hyperpolarized motoneurones. While potentiated by the uptake inhibitor nipecotic acid, the synaptic depressant effect of GABA was not antagonized by bicuculline. 7. The synaptic depressant effect of baclofen was neither blocked by GABAA antagonists bicuculline and picrotoxin (10-50 microM) nor by the GABAB antagonist phaclofen (0.1-1 mM). 8. It is suggested that baclofen depresses excitatory and inhibitory transmission in rat motoneurones by primarily a presynaptic mechanism in reducing the liberation of chemical transmitters from nerve endings via a phaclofen-insensitive GABAB receptor.

A comparison of fast and slow depolarizations evoked by 5-HT in guinea-pig coeliac ganglion cells in vitro.

1. 5-Hydroxytryptamine (5-HT) was applied by pressure ejection to coeliac ganglion cells of the guinea-pig maintained in vitro and responses measured intracellularly. 2. Cells responded in one of three ways to 5-HT: by (a) a fast, transient depolarization (43%), (b) a fast transient followed by a slow depolarization (biphasic response, 30%) or (c) a slow sustained depolarization (25%). 3. Fast depolarizations (response (a) above] were graded according to the duration of the ejection pulse. Maximal responses had a mean amplitude of 12 +/- 0.8 mV, a duration of 6.4 +/- 1.0 s, a latency of 0.4 +/- 0.1 s, were associated with a fall in membrane input resistance, increased in amplitude by hyperpolarization and probably mediated by an increased conductance to Na and K. The estimated reversal potential was -22.8 +/- 2.4 mV (n = 14). The maximal fast response seen in biphasically-responding cells (b) appeared similar to fast response (a). 4. Fast depolarizations (a) showed marked tachyphylaxis and were abolished by superfusion of the ganglion with 5-HT (100 microM). They were reduced in amplitude by tubocurarine (10-100 microM, pIC50 4.4), MDL 72222 (1-5 microM, pIC50 5.8), quipazine (1 microM reduced responses by 65 +/- 15%, n = 3), ICS 205-930 (1 microM reduced responses by 64 +/- 14%, n = 7) and metoclopramide (10 microM reduced responses by about 45%), but were unafected by methysergide (up to 1 microM) or hexamethonium (up to 1 mM). 5. Slow depolarizations (c) varied in amplitude with the duration of the ejection pulse. Maximal responses had a mean amplitude of 6.4 +/- 0.7 mV, a duration of 62 +/- 6 s, a latency of 3.5 +/- 0.8 s and were reduced in amplitude by methysergide (0.1-1 microM, pIC50 6.5) but not by MDL 72222 (1 microM). The maximal slow component in biphasically-responding cells (b) was similar in amplitude and duration to slow response (c), was partially blocked by methysergide (1-5 microM) in 4 of 6 cells and was enhanced by tubocurarine (50 microM) which reduced the fast component. 6. Slow depolarizations (b,c) were associated with either a small reduction or no change in membrane input resistance depending on the cell studied. Hyperpolarization had variable effects on slow depolarization amplitude. 7. It was concluded that the fast, phasic depolarization is mediated by an ionic mechanism and by receptors both of which are distinct from those involved in the slow depolarization. The receptor mediating the fast depolarization is a 5-HT3 receptor while that mediating the slow depolarization has yet to be identified.