Dynorphin A (1–13) Neurotoxicity in Vitro: Opioid and Non-Opioid Mechanisms in Mouse Spinal Cord Neurons

Dynorphin A is an endogenous opioid peptide that preferentially activates κ-opioid receptors and is antinociceptive at physiological concentrations. Levels of dynorphin A and a major metabolite, dynorphin A (1–13), increase significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both κ-opioid and N-methyl- -aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through κ-opioid and/or NMDA receptor types. Despite these findings, few studies have systematically explored dynorphin toxicity at the cellular level in defined populations of neurons coexpressing κ-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both κ-opioid and NMDA receptors from embryonic mouse spinal cord and examined the effects of dynorphin A (1–13) on intracellular calcium concentration ([Ca²⁺]_i) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. At micromolar concentrations, dynorphin A (1–13) elevated [Ca²⁺]_i and caused a significant loss of neurons. The excitotoxic effects were prevented by MK-801 (Dizocilpine) (10 μM), 2-amino-5-phosphopentanoic acid (100 μM), or 7-chlorokynurenic acid (100 μM)—suggesting that dynorphin A (1–13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (−)-naloxone (3 μM), or the more selective κ-opioid receptor antagonist nor-binaltorphimine (3 μM), exacerbated dynorphin A (1–13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 μM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 μM). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided indirect evidence that dynorphin also stimulates κ-opioid receptors and suggests that κ receptor activation may be moderately neuroprotective in the presence of an excitotoxic insult. Our findings suggest that dynorphin A (1–13) can have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, dynorphin A potentially modulates secondary neurodegeneration in the spinal cord through complex interactions involving multiple receptors and signaling pathways.     

Dynorphin B and spinal analgesia: induction of antinociception by the cannabinoids CP55,940, Δ-THC and anandamide

The endogenous opioid dynorphin B was evaluated for its role in cannabinoid-induced antinociception. Previous work in our laboratory has shown that the synthetic, bicyclic cannabinoid, CP55,940, induces the release of dynorphin B whilst the naturally occurring cannabinoid, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), releases dynorphin A. The dynorphins contribute in part to the antinociceptive effects of both cannabinoids at the level of the spinal cord. The present study compares dynorphin B released from perfused rat spinal cord in response to acute administration of anandamide (AEA), Δ⁹-THC and CP55,940 at two time points, 10 min and 30 min post administration, and attempts to correlate such release with antinociceptive effects of the drugs. Dynorphin B was collected from spinal perfusates of rats pretreated with Δ⁹-THC, CP55,940 or AEA. The supernatant was lyophilized and the concentrations of dynorphin B were measured via radioimmunoassay. At a peak time of antinociception (10 min), CP55,940 and Δ⁹-THC induced significant two-fold increases in the release of dynorphin B. AEA did not significantly release dynorphin B. Upon a 30-min pretreatment with the drugs, no significant dynorphin B release was observed, although antinociceptive effects persisted for CP55,940 and Δ⁹-THC. Previous work indicates that Δ⁹-THC releases dynorphin A while AEA releases no dynorphin A. This study confirms that although all three test drugs produced significant antinociception at 10 min, the endocannabinoid, AEA, does not induce antinociception via dynorphin release. Thus, our data indicate a distinct mechanism which underlies AEA-induced antinociception.     

Stimulation of hypothalamic β-endorphin and dynorphin release by corticotropin-releasing factor (in vitro)

Corticotropin-releasing factor (CRF) at doses of 10⁻¹²–10⁻⁸ M significantly stimulated the release of β-endorphin and dynorphin from superfused rat hypothalamic slices. These effects were shown to be mediated by the CRF receptor since they were antagonized by the CRF receptor antagonist α-helical CRF_9–41 (10⁻⁶ M). The two opioid peptides showed different time courses of response and in the case of β-endorphin, an attenuation of the response upon continued exposure to CRF was observed.     

The effect of intrathecal selective agonists of Y1 and Y2 neuropeptide Y receptors on the flexor reflex in normal and axotomized rats

We have examined the effects of intrathecal (i.t.) administration of [Leu³¹,Pro³⁴]-neuropeptide Y (NPY) or NPY-(13–36), selective agonists of NPY Y₁ or Y₂ receptors, respectively, on the excitability of the flexor reflex in normal rats and after unilateral transection of the sciatic nerve. In rats with intact and sectioned sciatic nerves, i.t. [Leu³¹,Pro³⁴]-NPY induced a similar biphasic effect on the flexor reflex with facilitation at low doses and facilitation followed by depression at high doses. In contrast, i.t. NPY-(13–36) only facilitated the flexor reflex in normal rats, and at high dose it caused ongoing discharges in the electromyogram. NPY-(13–36) caused dose-dependent depression of the flexor reflex in rats after sciatic nerve transection, in addition to its facilitatory effect. Topical application of [Leu³¹,Pro³⁴]-NPY or NPY-(13–36) caused a moderate and brief reduction in spinal cord blood flow. No difference was noted between the vasoconstrictive effect of [Leu³¹,Pro³⁴]-NPY and NPY-(13–36). It is suggested that activation of Y₁ receptors may be primarily responsible for the reflex depressive effect of i.t. neuropeptide Y in rats with intact sciatic nerves, whereas both Y₁ and Y₂ receptors may be involved in mediating the depressive effect of NPY after axotomy.     

Neuropharmacological characterization of local ibogaine effects on dopamine release

Summary Local perfusion with ibogaine (10^–6M—10^–3M) via microdialysis probes in the nucleus accumbens or striatum of rats produced a biphasic dose-response effect on extracellular dopamine levels. Lower doses (10^–6M—10^–4M) produced a decrease while higher doses (5 × 10^–4M—10^–3M) produced an increase in dopamine levels. Dihydroxyphenylacetic acid (DOPAC) levels were not effected. Naloxone (10^–6M) and norbinaltorphimine (10^–6M—10^–5M) did not affect dopamine levels, but when co-administered with ibogaine (10^–4M) blocked the decrease in dopamine levels produced by ibogaine. Ibogaine (10^–3M) stimulation of dopamine levels in the striatum was calcium independent and not blocked by tetrodotoxin (10^–5M). Pretreatment with cocaine (15mg/kg), reserpine (5mg/kg) or alpha-methyl-paratyrosine (250mg/kg) given intraperitoneally significantly reduced ibogaine (10^–3M) stimulation of striatal dopamine levels. In striatal synaptosomes, both ibogaine and harmaline (10^–7—10^–4M) produced dose-dependent inhibition of [³H]-dopamine uptake. These findings suggest that ibogaine has both inhibitory and stimulatory effects on dopamine release at the level of the nerve terminal. It is suggested that the inhibitory effect is mediated by kappa opiate receptors while the stimulatory effect is mediated by interaction with the dopamine uptake transporter.     

Des-tyrosine dynorphin A-(2–13) improves carbon monoxide-induced impairment of learning and memory in mice

The effects of des-tyrosine¹ dynorphin A-(2–13) (dynorphin A-(2–13)) on carbon monoxide (CO)-induced impairment of learning and memory in mice were investigated using a Y-maze task and a passive avoidance test. The lower percentage alternation and shorter step-down latency of the CO-exposed group indicated that learning and/or memory impairment occurred in mice 5 and 7 days after CO exposure, respectively. Administration of dynorphin A-(2–13) (1.5 and/or 5.0 nmol/mouse, intracerebroventricularly (i.c.v.)) 30 min before behavioral tests improved the CO-induced impairment in alternation performance and the CO-induced shortened step-down latency. We previously reported that dynorphin A-(1–13) improved the impairment of learning and/or memory via kappa opioid receptor mediated mechanisms. To determine whether the effect of dynorphin A-(2–13) was also mediated via kappa opioid receptors, we attempted to block its action using a selective kappa opioid receptor antagonist, nor-binaltorphimine (nor-BNI). Nor-BNI (4.9 nmol/mouse, i.c.v.) did not block the effects of dynorphin A-(2–13) on the CO-induced impairment of learning and/or memory. These results indicate that dynorphin A-(2–13) improves impairment of learning and/or memory via a non-opioid mechanism.     

Dynorphin A-(1–13) attenuates basal forebrain-lesion-induced amnesia in rats

The effects of dynorphin A-(1–13), an endogenous κ opioid agonist, on basal forebrain (BF)-lesion-induced amnesia in rats were investigated using step-through-type passive avoidance task. The BF was lesioned by injecting the cholinergic neurotoxin ibotenic acid (6 μg/side). The number of rats achieving the cut-off time (600 s) of step-through latency (STL) in BF-lesioned group significantly decreased as compared with that in sham-operated group. Dynorphin A-(1–13) (0.3 μg) significantly increased the number of rats achieving the cut-off time of STL in BF-lesioned rats. These results suggest that dynorphins play an improving role in the impairment of memory processes in BF-lesioned rats.     

Neurotensin — macrophage interaction: Specific binding and augmentation of phagocytosis

Neurotensin (NT) was found to bind to thioglycollate-elicited mouse peritoneal macrophages and to modulate their phagocytic capability. A Scatchard analysis of the binding curve of [³H]NT suggests the presence of two subclasses of binding sites having a.−a K_D of 0.9 nM (4800 sites per cell) and b.−a K_D of 28 nM (33500 sites per cell). NT as well as two of its partial sequences, NT(8–13) and NT(6–13) competed with [³H]NT for its binding whereas NT(1–10) was rather ineffective. [³H]NT was also competitively displaced by tuftsin, substance P (SP) and by SP (1–4). The K_I values estimated for all the above competitive inhibitors of [³H]NT binding (except for NT) suggest interaction with the relatively low affinity sites.NT exerts a biphasic effect on the phagocytic response of macrophages. At a concentration range of 10⁻¹⁴–10⁻⁹ M NT had a dose dependent augmenting effect on the phagocytic response (up to 2 fold), further increase in concentration (>10⁻⁹M) of NT resulted in a gradual decrease of the augmented response which disappears at 10⁻⁷ M NT. NT(8–13), NT(6–13) as well as NT(1–10) augment the phagocytic response of macrophages. However the maximal effect with these peptides was attained at about 10⁻⁷ M and stayed at the same level at concentrations up to 10⁻⁵ M. The phagocytosis augmenting dose-response curves of these peptides resembled that of tuftsin and SP, two unrelated peptides.It is suggested that NT-phagocyte interaction may be of relevance in the regulation of the functions of phagocytic cells.     

Intrathecal dynorphin A1–13 and dynorphin A3–13 reduce rat spinal cord blood flow by non-opioid mechanisms

Radiolabeled microspheres were used to examine the effects of paralytic intrathecal doses of dynorphin A (Dyn A_1–13) and Dyn A_3–13 on rat brain and spinal cord blood flows and cardiac output. Dyn A_1–13 produced significant dose-related reductions in blood flow to lumbosacral and thoracic spinal cord without altering cardiac output and blood flow to brain and cervical spinal cord. Naloxone failed to block these effects. Dyn A_3–13, which lacks opioid activity, also significantly reduced blood flow in lumbosacral spinal cord. Thus, the paralytic effects of Dyn A in the rat may involve reductions in spinal cord resulting from non-opioid actions of Dyn A.     

Analgesic actions of dynorphin A(1–13) antiserum in the rat brain stem

This study was performed to evaluate the effects of dynorphin A(1–13) antiserum when microinjected into an active hyperalgesic region within the rat brain stem. When administered within the dorsal posterior mesencephalic tegmentum (DPMT) of intact conscious rats, dynorphin A(1–13) antiserum produced rapid onset and persistent prolongation of a low intensity thermally evoked tail avoidance response (LITETAR). These analgesic actions of the dynorphin A(1–13) antiserum appeared to be dose dependent. These studies support previous hypotheses about the existence of tonically active brain stem opioid hyperalgesic processes. Further, the results provide indirect evidence for a potential role of brain stem dynorphin(s) in facilitating pain.     

Localization of aromatase and 5α-reductase to neuronal and non-neuronal cells in the fetal rat hypothalamus

Experiments were designed to identify the neural cell type(s) responsible for the aromatization and 5α-reduction of androgens in the rat hypothalamus. Primary cultures of fetal rat hypothalamic cells, which had enhanced neuronal morphology, were treated at various times after plating with kainic acid (KA), a neurotoxic agent which selectively destroys neuronal cells. Neuronal morphology was disrupted in a time (0–6 days)- and dose (10⁻⁴–10⁻² M)-dependent fashion after KA treatment, with no apparent change in the appearance of the flattened, underlying non-neuronal cells. KA treatment for 4 days decreased aromatization by 94% in a dose-dependent fashion (10⁻⁴–10⁻² M KA), while 5α-reduction declined by no more than 25%. A 6-day time course with 10⁻³ M KA showed a dramatic decline in aromatization and no alteration in 5α-reduction. In control experiments, substance P, a neuronal peptide, declined after KA treatment while the activity of glutamine synthetase, a glial enzyme, did not change. We conclude from these results that aromatase is localized primarily to neuronal cells in the hypothalamus while 5α-reductase is confined primarily to non-neuronal cells.     

Morphine and dynorphin(1–13) microinjected into the medial preoptic area and nucleus accumbens: effects on sexual behavior in male rats

The effects on sexual behavior of opiate receptor stimulation within A10 and A14 terminal areas were examined in the following experiments. Morphine (0.01–6 nmol) and dynorphin(1–13) (0.01–3 pmol) were microinjected into the medial preoptic area (MPOA). Morphine (10–100 pmol) and dynorphin (10–100 fmol) injected into the MPOA reduced both the latency to ejaculate and the number of intromissions triggering ejaculation. Morphine (6 nmol) produced a failure to resume copulating following the second ejaculation. Morphine (1–10 nmol) injected into the nucleus accumbens (ACC) shortened the latency to the first intromission and lengthened the second postejaculatory interval. Naloxone (3 mg/kg i.p.) reversed the effects of morphine on intromission latency and attenuated the lowering of ejaculatory threshold.     

Activation of the efferent system in the isolated frog labyrinth: Effects on the afferent EPSPs and spike discharge recorded from single fibers of the posterior nerve

Intra-axonal recordings were obtained from single afferent fibres of the posterior nerve in the isolated labyrinth of the frog (Rana esculenta). EPSPs spike discharge were recorded both at rest and during rotary stimulation of the canal. Electrical stimulation of either the distal end of the cut posterior nerve or of the central stumps of the anterior-horizontal nerves elicited a frequency-dependent inhibitory effect on the afferent discharge arising from the posterior canal. Denervation experiments revealed that inhibition is mediated by efferent fibres exhibiting a high degree of branching in the proximal part of the eight nerve. The inhibitory effect was selectively cancelled by (1)d-tubocurarine 10-⁶ M; (2) atropine 5 x 10-⁵ M;(3) acetylcholine or carbachol 10⁻⁴ M; (4) eserine 10⁻⁵ M. Inhibition is thus most likely to be sustained by the release of acetylcholine from the efferent nerve terminals. Experiments in which the ionic composition of the external medium was modified suggest that the transmitter acts mainly by opening the chloride ion channels of the hair cell membrane. In some units the same stimulation pattern evoked a consistent increase in both EPSP and spike discharge, instead of inhibition. Such facilitation was unaffected by drugs or ionic modifications which block the efferent synapse, but disappeared after denervation. Inhibition and facilitation, therefore, act as two control mechanisms which are able to modify substantially, at the first stage of processing, the sensory information which is sent to the vestibular second order neurones.     

Structure-activity analysis of dynorphin A toxicity in spinal cord neurons: intrinsic neurotoxicity of dynorphin A and its carboxyl-terminal, nonopioid metabolites

Dynorphin A [dynorphin A (1-17)] is an endogenous opioid peptide that is antinociceptive at physiological concentrations. Levels of dynorphin A increase markedly following spinal cord trauma and may contribute to secondary neurodegeneration. Both kappa opioid and N-methyl-d-aspartate (NMDA) receptor antagonists can modulate the effects of dynorphin, suggesting that dynorphin is acting through kappa opioid and/or NMDA receptor types. Despite these findings, few studies have critically examined the mechanisms of dynorphin A neurotoxicity at the cellular level. To better understand how dynorphin affects cell viability, structure-activity studies were performed examining the effects of dynorphin A and dynorphin A-derived peptide fragments on the survival of mouse spinal cord neurons coexpressing kappa opioid and NMDA receptors in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. Dynorphin A caused significant neuronal losses that were dependent on concentration (> or = 1 microM) and duration of exposure. Moreover, exposure to an equimolar concentration of dynorphin A fragments (100 microM) also caused a significant loss of neurons. The rank order of toxicity was dynorphin A (1-17) > dynorphin A (1-13) congruent with dynorphin A (2-13) congruent with dynorphin A (13-17) (least toxic) > dynorphin A (1-5) ([Leu(5)]-enkephalin) or dynorphin A (1-11). Dynorphin A (1-5) or dynorphin A (1-11) did not cause neuronal losses even following 96 h of continuous exposure, while dynorphin A (3-13), dynorphin A (6-17), and dynorphin A (13-17) were neurotoxic. The NMDA receptor antagonist MK-801 (dizocilpine) (10 microM) significantly attenuated the neurotoxic effects of dynorphin A and/or dynorphin-derived fragments except dynorphin A (13-17), suggesting that the neurotoxic effects of dynorphin were largely mediated by NMDA receptors. Thus, toxicity resides in the carboxyl-terminal portion of dynorphin A and this minimally includes dynorphin A (3-13) and (13-17). Our findings suggest that dynorphin A and/or its metabolites may contribute significantly to neurodegeneration during spinal cord injury and that alterations in dynorphin A biosynthesis, metabolism, and/or degradation may be important in determining injury outcome.     

Cells in the anteroventral cochlear nucleus are insensitive to l-glutamate and l-aspartate; excitatory synaptic responses are not blocked by d-α-aminoadipate

Auditory nerve fibers transmit signals from the cochlea to the 3 regions of the cochlear nuclear complex, the anteroventral (AVCN), posteroventral, and dorsal cochlear nucleus in the brainstem. It has been suggested that the amino acids l-aspartate and l-glutamate might serve as a neurotransmitter in auditory nerve fibers^{6–10,13,17–20}. The sensitivity of postsynaptic cells in the cochlear nuclei to these amino acids has been tested by iontophoretic techniques^4,9,10. One difficulty with these experiments is that responses were recorded only extracellularly. A second difficulty is that the concentrations needed to affect cells could not be determined. To avoid these difficulties a brain slice preparation was used to test the sensitivity of cells in the AVCN to bath applied l-glutamate and l-aspartate at concentrations ranging from 10⁻⁵ to 10⁻² M. All cells that were tested in the cochlear nuclear complex were insensitive at all concentrations used; the resting potentials and the input resistances remained unchanged and the synaptic responses to electrical stimulation of the auditory nerve were not desensitized. All cells that were tested in the hippocampus, however, depolarized in the presence of 10⁻⁴ M l-glutamate and l-aspartate. The synaptic responses to electrical stimulation of the auditory nerve were not blocked by d-α-aminoadipate, an amino acid which has been shown to block excitation of cells in the cochlear nuclei by auditory nerve fibers¹⁰. The results are not consistent with l-glutamate and l-aspartate serving as neurotransmitters in the AVCN.     

The muscarinic modulation of [H]

The ontogeny of the noradrenergic receptor subtypes modulating hypoglossal (XII) nerve inspiratory output was characterized. Noradrenergic agents were locally applied over the XII nucleus of rhythmically active medullary slice preparations isolated from mice between zero and 13 days of age (P0–P13) and the effects on XII inspiratory burst amplitude quantified. The α₁ receptor agonist phenylephrine (PE, 0.1–10 μM) produced a dose-dependent, prazosin-sensitive (0.1–10 μM) increase in XII nerve inspiratory burst amplitude. The magnitude of this potentiation increased steadily from a maximum of 15±8% in P0 mice to 134±4% in P12–P13 mice. The β receptor agonist isoproterenol (0.01–1.0 mM) produced a prazosin-insensitive, propranolol-sensitive potentiation of XII nerve burst amplitude. The isoproterenol-mediated potentiation increased with development from 27±5% in P0–P1 slices, to 37±3% in P3 slices and 45±4% in P9–P10 slices. The α₂ receptor agonist clonidine (1 mM) reduced XII nerve inspiratory burst amplitude in P0–P3 slices by 29±5%, but had no effect on output from P12–P13 slices. An α₂ receptor-mediated inhibition of inspiratory activity in neonates (P0–P3) was further supported by a 19±3% reduction in XII nerve burst amplitude when norepinephrine (NE, 100 μM) was applied in the presence of prazosin (10 μM) and propranolol (100 μM). Results indicate that developmental increases in potentiating α₁ and, to a lesser extent, β receptor mechanisms combine with a developmentally decreasing inhibitory mechanism, most likely mediated by α₂ receptors, to determine the ontogenetic time course by which NE modulates XII MN inspiratory activity.     

Developmental modulation of mouse hypoglossal nerve inspiratory output in vitro by noradrenergic receptor agonists

The ontogeny of the noradrenergic receptor subtypes modulating hypoglossal (XII) nerve inspiratory output was characterized. Noradrenergic agents were locally applied over the XII nucleus of rhythmically active medullary slice preparations isolated from mice between zero and 13 days of age (P0–P13) and the effects on XII inspiratory burst amplitude quantified. The α₁ receptor agonist phenylephrine (PE, 0.1–10 μM) produced a dose-dependent, prazosin-sensitive (0.1–10 μM) increase in XII nerve inspiratory burst amplitude. The magnitude of this potentiation increased steadily from a maximum of 15±8% in P0 mice to 134±4% in P12–P13 mice. The β receptor agonist isoproterenol (0.01–1.0 mM) produced a prazosin-insensitive, propranolol-sensitive potentiation of XII nerve burst amplitude. The isoproterenol-mediated potentiation increased with development from 27±5% in P0–P1 slices, to 37±3% in P3 slices and 45±4% in P9–P10 slices. The α₂ receptor agonist clonidine (1 mM) reduced XII nerve inspiratory burst amplitude in P0–P3 slices by 29±5%, but had no effect on output from P12–P13 slices. An α₂ receptor-mediated inhibition of inspiratory activity in neonates (P0–P3) was further supported by a 19±3% reduction in XII nerve burst amplitude when norepinephrine (NE, 100 μM) was applied in the presence of prazosin (10 μM) and propranolol (100 μM). Results indicate that developmental increases in potentiating α₁ and, to a lesser extent, β receptor mechanisms combine with a developmentally decreasing inhibitory mechanism, most likely mediated by α₂ receptors, to determine the ontogenetic time course by which NE modulates XII MN inspiratory activity.     

A carrier for GABA uptake exists on noradrenaline nerve endings in selective rat brain areas but not on serotonin terminals

Summary -Aminobutyric acid (GABA) increased in a concentration-dependent way (3–300M) the basal release of tritium from rat cerebral cortex and hippocampus synaptosomes, prelabelled with³H-noradrenaline (³H-NA); however, GABA was ineffective on hypothalamic nerve endings. The effect displayed by low concentrations (<10M) of GABA was largely bicuculline-sensitive. Muscimol mimicked GABA, while (–)baclofen was inactive.The releasing effects produced by concentrations of GABA higher than 10M were largely prevented by SK&F89976A, SK&F 100330A and SK&F 100561, three novel GABA uptake inhibitors. When present together, GABA uptake blocker and bicuculline counteracted entirely the GABA effects. The basal release of³H-5-hydroxytryptamine (³H-5-HT) in synaptosomes from various CNS regions was not affected by GABA. In conclusion: GABA can enhance³H-NA release not only through GABA-A receptors but also by penetrating into NA terminals through a GABA uptake system. This implies coexistence of carriers for NA and GABA uptake on a same nerve terminal. The carrier coexistence occurs in selective CNS areas. The phenomenon appears to be transmitter-selective.     

Vasoactive intestinal peptide stimulates melatonin release from perifused pineal glands of rats

Summary The rat pineal gland is known to release melatonin in response to noradrenergic stimulation. The effect of vasoactive intestinal peptide (VIP), one of the neuropeptides present in the pineal, was examined on perifused rat pineal glands. VIP stimulated melatonin release with a dose-dependent effect above 10^–7 M. In regard of kinetic characteristics, the pattern of melatonin release after VIP stimulation was similar to that after isoproterenol stimulation. 10^–6 M VIP-stimulated melatonin release was not altered when the pineal glands were treated with 10^–5 M propranolol (a -adrenergic antagonist) or 10^–5 M prazosin (an ₁-adrenergic antagonist). Thus VIP has a noradrenergic-independent effect on melatonin secretion. Conversely, this VIP effect is greatly inhibited by the specific action of a VIPergic antagonist. This suggests that VIP acts on melatonin synthesis through its own binding sites.This study demonstrates that melatonin secretion from rat pineal glands may be elicited through a VIPergic system which is independent of the well-known noradrenergic system.     

Anatomical evidence for two spinal ‘afferent–interneuron–efferent’ reflex pathways involved in micturition in the rat: a ‘pelvic nerve’ reflex pathway and a ‘sacrolumbar intersegmental’ reflex pathway

We labeled interneurons in the L1–L2 and L6–S1 spinal cord segments of the rat that are involved in bladder innervation using transneuronal retrograde transport of pseudorabies virus (PRV) in normal animals and in animals with selected nerve transections. Preganglionic neurons were identified using antisera against choline acetyltransferase (ChAT). In some experiments we labelled parasympathetic preganglionic neurons (PPNs) in the L6–S1 spinal cord by retrograde transport of Fluorogold from the major pelvic ganglion. We identified bladder afferent terminals using the transganglionic transport of the anterograde tracer cholera toxin subunit b. We present anatomical evidence for two spinal pathways involved in innervation of the bladder. First, in the intact rat, afferent information from the bladder connects, via interneurons in L6–S1, to the PPNs that provide the efferent innervation of the bladder. The afferent terminals were located mainly in close apposition to interneurons located dorsal to the retrogradely labeled PPNs. Second, using L6–S1 ganglionectomies or L6–S1 ventral root rhizotomies we limited viral transport to the sympathetic pathways innervating the bladder. This procedure also labelled interneurons (but not PPNs) with PRV in the L6–S1 spinal cord in a location very similar to those described in the intact rat. These interneurons also receive bladder afferent terminals but we propose that they project to sympathetic preganglionic neurons, most of which are in the L1–L2 spinal segments. Based on this anatomical evidence, we propose the existence of two spinal reflex pathways involved in micturition: a pathway limited to a reflex arc in the pelvic nerve (presumably excitatory to the detrusor muscle); and a pathway involving the pelvic nerve and sympathetic nerve fibers, some of which may travel in the hypogastric (presumably inhibitory to the detrusor muscle).