The role of spinal cord 5-HT1A and 5-HT1B receptors in the modulation of a spinal nociceptive reflex

The role of the 5-hydroxytryptamine (5-HT) receptor subtypes in the spinal cord in the regulation of nociception is unknown. This study examined whether administration of different 5-HT₁ receptor agonists into the spinal subarachnoid space of mice modulates the nociceptive tail-flick reflex, and whether effects on the tail-flick reflex involve changes in tail skin temperature. The tail-flick latencies (the time needed to evoke the tail-flick reflex by noxious radiant heat) were significantly increased after intrathecal (i. th.) injection of 5-HT (10–20 μg), the 5-HT_1A/5-HT_1B receptor agonist5-methoxy-N,N-dimethyltryptamine (5-MeODMT, 10–20 μg), the selective 5-HT_1A receptor agonist8-hydroxy-2-di-n-propylamino)tetralin (8-OH-DPAT, 20 μg), and after i.th. injection of1(m-chlorophenyl)piperazine (mCPP, 5–20 μg) and5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969, 5–20 μg) which have high affinity for the 5-HT_1B receptors. None of the 5-HT₁ receptor agonists had the ability to change the tail skin temperature. The results show that in the mouse i.th. injection of both 5-HT_1A and 5-HT_1B receptor agonists has the ability to inhibit the tail-flick reflex without interfering with the tail skin temperature.     

The role of spinal cord 5-HT1A and 5-HT1B receptors in the modulation of a spinal nociceptive reflex

The role of the 5-hydroxytryptamine (5-HT) receptor subtypes in the spinal cord in the regulation of nociception is unknown. This study examined whether administration of different 5-HT1 receptor agonists into the spinal subarachnoid space of mice modulates the nociceptive tail-flick reflex, and whether effects on the tail-flick reflex involve changes in tail skin temperature. The tail-flick latencies (the time needed to evoke the tail-flick reflex by noxious radiant heat) were significantly increased after intrathecal (i. th.) injection of 5-HT (10-20 micrograms), the 5-HT1A/5-HT1B receptor agonist 5-methoxy-N,N-dimethyltryptamine (5-MeODMT, 10-20 micrograms), the selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 20 micrograms) and after i.th. injection of 1(m-chlorophenyl)piperazine (mCPP, 5-20 micrograms) and 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969, 5-20 micrograms) which have high affinity for the 5-HT1B receptors. None of the 5-HT1 receptor agonists had the ability to change the tail skin temperature. The results show that in the mouse i.th. injection of both 5-HT1A and 5-HT1B receptor agonists has the ability to inhibit the tail-flick reflex without interfering with the tail skin temperature.     

Functional evidence for the rapid desensitization of 5-HT3 receptors on vagal afferents mediating the Bezold–Jarisch reflex

The aim of this study was to determine whether 5-hydroxytryptamine (5-HT)₃ receptors on cardiopulmonary afferents mediating the Bezold–Jarisch reflex (BJR) desensitize upon repeated exposure to selective agonists. BJR-mediated falls in heart rate, diastolic arterial blood pressure and cardiac output elicited by the 5-HT₃-receptor agonists, phenylbiguanide (100 μg/kg, i.v.) or 2-methyl-5-HT (100 μg/kg, i.v.), progressively diminished upon repeated injection in conscious rats. The BJR responses elicited by 5-HT (40 μg/kg, i.v.) were markedly reduced in rats which had received the above injections of phenylbiguanide or 2-methyl-5-HT whereas the BJR responses elicited by -S-nitrosocysteine (10 μmol/kg, i.v.) were similar before and after the injections of the 5-HT₃ receptor agonists. These findings suggest that tachyphylaxis to 5-HT₃ receptor agonists may be due to the desensitization of 5-HT₃ receptors on cardiopulmonary afferents rather than the impairment of the central or peripheral processing of the BJR.     

Tachykinins protect cholinergic neurons from quinolinic acid excitotoxicity in striatal cultures

The neuroprotective effect of tachykinins against excitotoxic death of cholinergic neurons was studied in rat striatal cell cultures. Quinolinic acid (QUIN) and kainic acid (KA) produced a dose dependent decrease in choline acetyttransferase activity, but KA was more potent. Our results show that substance P (SP) totally reversed the toxicity induced by 125 μM QUIN but not by 40 μM KA. This effect was also observed using protease inhibitors or a SP-analog resistant to degradation, [Sar⁹]-Substance P. The survival of neuron specific enolase- and acetylcholinesterase (AChE)-positive cells after treatment with QUIN alone or in the presence of SP was also examined. We observed that, while a decrease in total cell number produced by QUIN was not prevented by SP treatment, AChE-positive cells were rescued from the toxic damage. To characterize the SP protective effect we used more selective agonists of the three classes of neurokinin (NK) receptors. [Sar⁹, Met(O₂)¹¹]-Substance P (NK₁ receptor agonist), [Nle¹⁰]-Neurokinin A (NK₂ receptor agonist) or [Me-Phe⁷]-Neurokinin B (NK₃ receptor agonist) were all able to block the toxic effect of QUIN on cholinergic activity. These results show that tachykinins provide an important protective support for striatal neurons, suggesting a possible therapeutical benefit in neurodegenerative disorders affecting cholinergic neurons.     

Pretreatment with SR48692 has different effects on central neurotensin-induced gastric mucosal defense and inhibition of gastric acid secretion in rats

Neurotensin is a tridecapeptide present in the brain and gastrointestinal tract. Administration of neurotensin into the brain results in responses in the gastrointestinal tract, suggesting a role for neurotensin in the interrelationships that comprise the brain–gut axis. Intracerebroventricular (i.c.v.) administration of neurotensin protects the gastric mucosa against injury caused by cold water restraint (CWR) and also inhibits gastrin-stimulated gastric acid secretion. The hypothesis tested was that these two actions of neurotensin are mediated via its high-affinity receptor. Rats were given neurotensin (60 μg, i.c.v.) prior to CWR or pylorus ligation after pretreatment with SR48692, a nonpeptide antagonist of the high-affinity neurotensin receptor (0.25 or 2.5 μg, i.c.v., or 10, 100, or 500 μg kg⁻¹, i.p.). Neurotensin reduced cold water restraint (CWR)-induced gastric mucosal injury and inhibited gastrin-stimulated acid secretion. Pretreatment with SR48692 (2.5 μg, i.c.v., or 100 μg kg⁻¹, i.p.) prior to CWR blocked neurotensin's protection of the gastric mucosa against injury. In contrast, pretreatment with 2.5 μg SR48692, i.c.v., did not block neurotensin-induced inhibition of acid secretion, whereas 500 μg kg⁻¹, i.p., partially blocked the inhibition. SR48692 (2.5 μg, i.c.v.) inhibited acid secretion, suggesting that SR48692 has agonist activity in this system. These results suggest that central neurotensin protects the gastric mucosa against CWR-induced injury via its high-affinity receptor. The receptor that mediates central neurotensin-induced inhibition of gastric acid secretion does not appear to be the high-affinity receptor since the neurotensin receptor antagonist SR48692, when given i.c.v., had agonist activity, inhibiting stimulated acid secretion. High-affinity neurotensin receptors in the periphery appear to play a role in inhibition of stimulated gastric acid secretion.     

F11 neuroblastoma × DRG neuron hybrid cells express inhibitory μ- and δ-opioid receptors which increase voltage-dependent K currents upon activation

The F11 cell line is a fusion product of cells of mouse neuroblastoma cell line N18TG-2 with embryonic rat dorsal-root ganglion (DRG) neurons. Previous biochemical results suggest that they express μ- and δ-opioid receptors that are negatively coupled to adenylate cyclase. The present study provides direct agonist-binding and electrophysiologic evidence of μ and δ, but not κ, receptor expression in F11 cells. Radioligand binding assaysshow that F11 cell membranes bind the μ- and δ-opioid receptor agonists, DAGO and DPDPE with K_d = 4.5 and 4.9 nM and B_max = 111 and 195 fmol/mg, respectively. Tight-seal patch-clamp recordings of F11 cells after several days in a differentiating culture medium (low serum, cyclic AMP and nerve growth factor) showed that: (i) the outward K⁺ current during pulsed depolarization in most of these cells was increased by either DAGO or DPDPE, but none were responsive to both opioids or to the κ-opioid receptor agonist, U-50,488H. The response was blocked by relevant receptor antagonists, naloxone, ß-funaltrexamine or naltrindole; (ii) cells without processes responded neither to DAGO nor to DPDPE; (iii) treatment with pertussis toxin blocked all opioid-induced increases in outward K⁺ current. The opioid-induced increase in voltage-dependent membrane K⁺ current in F11 cells resembles the inhibitory effect elicited by μ- and δ-opioid agonists in primary cultures of mouse DRG neurons.     

Locomotor suppressive and anxiolytic-like effects of urocortin 3, a highly selective type 2 corticotropin-releasing factor agonist

Murine urocortin 3 (mUcn 3), a member of the corticotropin releasing factor (CRF) peptide family, was recently identified. Of known agonists, this neuropeptide displays the highest degree of selectivity in binding to the CRF₂ receptor. These experiments sought to test the hypothesis that CRF₂ receptors have a role in modulating stress by examining intracerebroventricular (i.c.v.) administration of mUcn 3 in animal models of activation and anxiety. To investigate the effects of mUcn 3 on motor activity, rats were injected with mUcn 3 (0, 0.1, 1.0 or 10 μg, i.c.v.) 10 min prior to testing and activity was monitored for 6 h. To determine changes in novelty-induced exploration, rats were injected with mUcn 3 (0, 0.1, 1.0 or 10 μg, i.c.v.) 10 min prior to testing and examined in the elevated plus maze. Finally, delayed effects of mUcn 3 were tested in rats injected with 1.0 μg of mUcn 3 or vehicle 30 or 60 min prior to testing in the elevated plus maze. Injections of mUcn 3 significantly decreased locomotor activity in rats during the first hour of testing. In the elevated plus maze, mUcn 3 injections significantly increased open arm preference compared to vehicle when tested using a 10-min pretreatment interval. mUcn treated rats tested in the elevated plus maze following the delayed pretreatment interval did not differ from controls. These data demonstrate that injection of mUcn 3 leads to acute locomotor suppressive effects and decreases in stress-like behaviors, indicating an anxiolytic-like action for mUcn 3, and suggests a possible role of the CRF₂ receptor in the regulation of stress-related behavior.     

The peripheral nociceptive actions of intravenously administered 5-HT in the rat requires dual activation of both 5-HT2 and 5-HT3 receptor subtypes

In 16-week-old Sprague-Dawley rats lightly anesthetized with pentobarbital, 5-HT (3–96 μg/kg, i.v.;n = 6) produced distinct pseudaffective responses and a dose-dependent (slope= 17.2 ± 6.8s/log₁₀dose) inhibition of the tail-flick (TF) reflex (ED₅₀ = 32.6 ± 9.2 μg/kg). In the same rats, a 1:1 combination of α-methyl 5-HT (a 5-HT₂ receptor selective agonist) and 2-methyl 5-HT (a 5-HT₃ receptor selective agonist) (3–192 μg/kg, i.v.), produced the same profile of pseudaffective responses and also resulted in a dose-dependent (slope= 34.0± 7.0s/log₂dose) inhibition of the TF reflex (ED₅₀ = 88.4 ± 20.5 μg/kg). In contrast, administration of α-methyl 5-HT (3–192 μg/kg, i.v.) or 2-methyl 5-HT (3–192 μg/kg, i.v.) alone did not produce any pseudaffective responses or any change in TF latency from baseline. In conscious 16-week-old male Sprague-Dawley rats, administration of 5-HT (48 μg/kg, i.v.;n = 5), or a 1:1 combination of α-methyl 5-HT and 2-methyl 5-HT (total dose= 120 μg/kg, i.v.;mn = 5), resulted in a passive avoidance behavior assessed in a step-down paradigm (slopes= 139.7 ± 58.2and154.9 ± 63.9s/trial, respectively), and the same profile of distinct pseudaffective responses exhibited by the lightly pentobarbital-anesthetized rats. However, administration of either α-methyl 5-HT (96 μg/kg, i.v.;n = 4) or 2-methyl 5-HT (96 μg/kg, i.v.;n = 4), while producing significant 5-HT receptor-mediated cardiovascular responses, produced a learned behavior not different from saline (0.25 ml, i.v.;n = 6) (slopes= 7.6 ± 2.5, 6.3 ± 1.8and7.4 ± 3.6s/trial, respectively). These results are consistent with the hypothesis that the peripheral nociceptive responses to i.v. 5-HT requires dual activation of 5-HT₂ and 5-HT₃ receptor subtypes.     

5-HT inhibits lateral entorhinal cortical neurons of the rat in vitro by activation of potassium channel-coupled 5-HT1A receptors

Serotonin (1–40 μM) reduced input resistance by 20.6±6% and hyperpolarized stellate and pyramidal neurons of layers two and three of the lateral entorhinal cortex. 5-Carboxamidotryptamine, a 5-HT₁ agonist, and the selective 5-HT_1A agonist 8-hydroxy-dipropylaminotetralin mimicked the action of serotonin. The reversal potential of 5-HT-mediated hyperpolarizations was sensitive to the extracellular K⁺ concentration, indicating a potassium conductance change. Serotonin treatment suppressed excitatory amino acid-mediated synaptic potentials (by 48%, K_d=6.9 μM) and responses to exogenously applied glutamate (70.1±17% of control, n=7), but did not alter paired-pulse facilitation, indicating a postsynaptic site of action. Intracellular application of QX-314, a blocker of potassium conductance, significantly reduced depression of synaptic potentials by 5-HT agonists. In cells filled with QX-314, responses to exogenously applied glutamate were not reduced by serotonin or 5-carboxamidotryptamine application. These results indicate that the observed conductance increase associated with 5-HT application accounts for most if not all of the observed depressant effects of 5-HT_1A agonists on excitatory amino acid-mediated neurotransmission.     

General, μ and κ opioid antagonists in the nucleus accumbens alter food intake under deprivation, glucoprivic and palatable conditions

Ventricular microinjection studies found that whereas μ (β-funaltrexamine, B-FNA), μ₁ (naloxonazine) and κ (nor-binaltorphamine, Nor-BNI) opioid receptor antagonists, but not δ antagonists, reduce deprivation-induced intake, κ and μ, but not μ₁ or δ antagonists reduce both 2-deoxy- d-glucose (2DG) hyperphagia and sucrose intake. Since opioid agonists stimulate spontaneous food intake in the accumbens, the present study examined whether administration of either naltrexone, B-FNA or Nor-BNI in the accumbens altered intake under deprivation (24 h), glucoprivic (2DG: 500 mg/kg, i.p.) or palatable sucrose (10%) conditions. Naloxonazine's effects in the accumbens were also evaluated for deprivation-induced intake. Deprivation-induced intake was significantly decreased over 4 h by naltrexone (5–20 μg, 44%), B-FNA (1–4 μg, 55%) and Nor-BNI (4 μg, 31%), but not naloxonazine (10 μg) in the accumbens. 2DG hyperphagia was significantly decreased by naltrexone (10–20 μg, 79%), B-FNA (1–4 μg, 100%) and Nor-BNI (1–4 μg, 75%) in the accumbens. Sucrose intake was significantly decreased by naltrexone (50 μg, 27%) and B-FNA (1–4 μg, 37%), but not Nor-BNI in the accumbens. These data suggest that μ receptors, and particularly the μ₂ binding site in the accumbens are responsile for the opioid modulation of these forms of intake in this nucleus, and that this control may be acting upon the amount of intake per se.     

Pharmacological characterization of the ameliorating effect on short-term memory impairment and antinociceptive effect of KT-90 in mice

(−)-3-Acetyl-6β-acetylthio-N-cyclopropylmethyl-normorphine (KT-90) is a synthesized compound that binds to μ-, δ- and κ-opioid receptors in vitro. KT-90 induces analgesia in the tail-flick test and this effect is antagonized by nor-BNI, a selective κ-opioid receptor antagonist. However, lower doses of KT-90 antagonize morphine-induced analgesia. We reported that κ-opioid receptor agonists such as U-50,488H and dynorphin A (1-13), improved scopolamine-induced impairment of learning and memory in mice and/or rats. In this study, the effects of KT-90 were investigated in an acetic acid-induced writhing test and scopolamine-induced memory impairment test using spontaneous alternation performance in a Y-maze. Male ddY mice were treated with scopolamine (1.65 μmol/kg, s.c.) 30 min before the behavioral test. KT-90 (0.07–2.35 μmol/kg, s.c.) was injected 30 min before testing. In the writhing test, the antinociceptive effect of KT-90 (0.71 μmol/kg) was completely antagonized by a selective μ-opioid receptor antagonist, β-funaltrexamine (10.2 nmol/mouse, i.c.v.) and partially antagonized by nor-BNI (4.9 nmol/mouse, i.c.v.), but it was not antagonized by a selective δ-opioid receptor antagonist, naltrindole (9.1 pmol/mouse, i.c.v.). KT-90 significantly improved the impairment of spontaneous alternation induced by scopolamine. The ameliorating effect of KT-90 was not antagonized by nor-BNI, but was almost completely antagonized by a selective σ receptor antagonist, NE-100 (2.6 μmol/kg, i.p.). These results suggested that the KT-90-induced antinociceptive effect was mediated by μ- and partially by κ-opioid receptors, and the KT-90-induced improvement in scopolamine-induced impairment of spontaneous alternation was mediated mainly via σ receptors.     

Cardiovascular interactions of nonivamide, glyceryl nonivamide, capsaicin analogues, and substance P antagonist in rats

Nonanoyl vanillylamide-4-o-glycerol (glyceryl nonivamide, GLNVA) a nonpungent ether-linked derivative of nonanoyl vanillylamide (nonivamide, NVA) was compared to capsaicin (CAP) and NVA with regard to its depressor response in rats. IV injection of CAP and NVA (10−1 to 10⁻⁴ mg/kg) in Wistar rats elicit a triphasic blood pressure response, bradycardia, and aponea. However, IV injection of GLNVA results in a monophasic reduction in blood pressure, with little effect on heart rate and respiration. The depressor response to GLNVA was not diminished by bilateral vagotomy or by systemic pretreatment with atropine. Following the CAP pretreatment, the delayed hypotension induced by CAP, NVA, and the hypotension of GLNVA was almost abolished. Injection of CAP, NVA (10 μg/kg), or GLNVA (100 μg/kg) into one femoral artery elicited a fall in blood pressure in the rat. This effect was abolished following intrathecal injection of substance P antagonist [D-Pro², D-Trp^7,9]- SP. Microejections of CAP, NVA, or GLNVA into the nucleus tractus solitarii (NTS) evoked hypotension, the bradycardia following microejection of CAP and NVA into the NTS occurred only at higher doses of GLNVA. From these results it is suggested that GLNVA appears to act more exclusively than CAP by stimulating peripheral perivascular small diameter C-fiber sensory nerves.     

Differential serotonergic inhibition of in vitro striatal [3H]acetylcholine release in prenatally cocaine-exposed male and female rats

Previous research indicates that prenatal cocaine (pCOC)-exposure results in greater 5-HT₃ agonist-induced inhibition of electrically evoked [³H]acetylcholine (ACh) overflow in rat striatal slices. The present study examines the effects of fluoxetine (FLU)-induced and exogenous serotonin (5-HT) on electrically evoked ACh release from striatal slices prepared from adult male and female (in periods of diestrus or proestrus) rats exposed to saline or cocaine in utero. Additionally, we assessed the impact of monoaminergic receptor stimulation on evoked ACh release by superfusion with selective 5-HT₂, 5-HT₃ and D₂ receptor antagonists in the presence of FLU-induced and exogenous 5-HT and measuring the capacity of these drugs to reverse inhibitory effects of 5-HT. Given our previous findings of accentuated inhibition of ACh release by 5-HT₃ agonism in striata of pCOC-exposed adult rats, we hypothesized that superfusion of endogenous and exogenous 5-HT would lead to greater suppression of evoked ACh release in this group of animals. Our results indicated that ACh release from slices of all prenatal saline (pSAL) rats was inhibited comparably by FLU (10 μM)-elicited increases in endogenous 5-HT or by increases elicited with application of exogenous 5-HT (5 μM). Robust FLU-mediated inhibition of ACh release was evident in slices from pCOC male and pCOC diestrus female rats vs. their respective PSAL control groups. Superfusion of striatal slices with 5-HT (5 μM) produced a pattern of ACh inhibition similar to that produced by FLU; however, the magnitude of ACh inhibition was consistently greater than that observed with FLU. Inhibition of ACh overflow by FLU was blocked by co-superfusion with ketanserin, a 5-HT₂ receptor antagonist, ICS-205,930, a 5-HT₃ receptor antagonist or sulpiride, a D₂ receptor antagonist. Conversely, serotonergic inhibition of ACh overflow was only blocked by a high concentration of ICS-205,930 (5 μM) and was completely reversed by sulpiride (1 μM). Collectively, these findings demonstrate serotonergic modulation of cholinergic neurons varying as a function of prenatal treatment, sex and, for females, phase of estrous. Inhibition of ACh release by 5-HT appears to be mediated by a complex relationship between 5-HT₂, 5-HT₃ and D₂ receptor regulation, as the blockade of any of these receptors reversed the inhibitory effects of FLU on ACh release. Conversely, in the case of exogenous 5-HT-induced inhibition, only blockade of D₂ receptors and high concentrations of the 5-HT₃ receptor antagonists were capable of reversing monoaminergic inhibition. These data support the hypothesis that the enhanced serotonergic modulation of ACh neurons in pCOC-exposed animals is largely mediated by dopamine (DA) and reflect a major biochemical persistence of neurodevelopmental adaptations elicited by early cocaine exposure.     

Relationship of increased food intake and plasma ACTH levels to 5-HT1A receptor activation in rats

Various putative agonists of the 5-HT_1A receptor subtype induce feeding in rats, probably by activating raphé somatodendritic 5-HT autoreceptors. These drugs also produce a marked increase in plasma concentrations of corticotropin (ACTH). In the present experiment we attempted to localize the site of action of 5-HT_1A agonists on the secretion of ACTH and examined the relationship between 5-HT_1A agonist-induced feeding and ACTH secretion. Rats were injected with either the high affinity 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (0.016-1.0 mg/kg, s.c.) or the novel anxiolytics buspirone, gepirone or ipsapirone (2.0–16.0 mg/k/g, s.c.), and either had their food intake measured 2 hr post injection or were sacrificed 30–40 min post injection for measurement of plasma ACTH. Plasma ACTH also was measured in rats pretreated with the serotonin synthesis inhibitor, para-chlorophenylalanine (PCPA) for three days (150 mg/kg, i.p. per day) and subsequently injected with 8-OH-DPAT (0.3 mg/kg, s.c.).

As previously reported, the 5-HT_1A agonists increased both food agonists increased both food intake and plasma ACTH concentrations. After 8-OH-DPAT, ipsapirone and gepirone the amount of food consumed was positively correlated with the concentration of plasma ACTH. No such correlation was evident following buspirone. PCPA pretreatment resulted in near total depletion of brain 5-HT content but had no effect on the ACTH rise induced by 8-OH-DPAT. Therefore, in contrast to the presynaptic site previously proposed for 5-HT_1A agonist-induced feeding, the present results suggest a agonist-induced feeding, the present results suggest a postsynaptic location for the 5-HT_1A receptor mediating ACTH release.     

Excitatory and indirect inhibitory actions of 5-hydroxytryptamine on sympathetic preganglionic neurones in the neonate rat spinal cord in vitro

The action of 5-hydroxytryptamine (5-HT) on sympathetic preganglionic neurones (SPN) was studied by intracellular recordings in thin slices of neonatal rat spinal cord in vitro. Superfusion of 5-HT (1–270 μM) to SPN caused a concentration dependent slow depolarization or inward current and an increase in synaptic activity consisting of both EPSPs and IPSPs. The slow depolarization was still present after superfusion with TTX. Similar effects were seen during superfusion with 5-carboxamidotryptamine (5-CT) or -methyl-5-hydroxytryptamine (-me-5-HT). A comparison with the potency of 5-HT was made for 5-CT or -me-5-HT on the same neurone by determining the magnitude of the slow depolarization to different concentrations of agonist. This showed that the apparent potency of the agonists was 5-CT> 5-HT> -me-5-HT even in the presence of fluoxetine, a 5-HT uptake inhibitor. The 5-HT-induced slow depolarization was partially blocked by ketanserin but full recovery was not observed. The results suggest that the excitatory action of 5-HT on SPN is mediated via an atypical 5-HT₂ receptor or a 5-HT_1C-like receptor. The 5-HT-induced IPSPs were reversibly blocked by superfusion with strychnine, suggesting they were mediated by glycine.     

Effects of veratridine and high potassium on micro-opioid receptor internalization in the rat spinal cord: stimulation of opioid release versus inhibition of internalization

Veratridine and high K⁺-induced μ-opioid receptor (MOR) internalization in rat spinal cord slices by evoking opioid release. Veratridine induced up to 75% MOR internalization but showed an atypical concentration–response: its effect increased steeply from 5 μM to 10 μM, and declined thereafter to disappear at 100 μM. At 100 μM, veratridine also inhibited of MOR internalization induced by exogenous endomorphin-2. This inhibition was caused by Na⁺ entry, since the Na⁺ ionophore monensin (50 μM) also inhibited endomorphin-induced MOR internalization. In contrast, veratridine induced neurokinin 1 receptor internalization (by evoking substance P release) without any inhibition at high concentrations. KCl evoked up to 80% MOR internalization, which disappeared in the presence of lidocaine or in the absence of peptidase inhibitors, indicating that it involved neuronal firing and peptide release. Unlike veratridine, KCl did not inhibit MOR internalization at high concentrations. However, both KCl and veratridine evoked more MOR internalization when applied for 2 min than for 20 min because of a direct inhibition of MOR internalization with the longer incubation times. These results show that short incubations with 20 μM veratridine or 30 mM KCl are optimal stimuli to evoke opioid release and MOR internalization in the spinal cord.     

Opiate antagonists inhibit feeding induced by 8-OH-DPAT: possible mediation in the nucleus accumbens

Previous work has shown that the 5-hydroxytryptamine (5-HT)_1A receptor agonist8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) elicits a variety of behaviours, including feeding in rats. These effects are accompanied by reduced 5-HT neurotransmission resulting from activation of somatodendritic 5-HT receptors located in the midbrain raphe nuclei. Dopamine antagonists injected either peripherally or into the nucleus accumbens reverse 8-OH-DPAT-induced feeding. Thus a facilitation of dopamine activity, secondary to reduced 5-HT activity, may be involved in mediating 8-OH-DPAT-induced feeding. Opiate antagonists have been shown previously to reduce several dopamine-dependent behaviours including feeding induced by dopaminergic drugs, tail pinch and electrical brain stimulation. Therefore experiments were conducted to assess the effects of opiate antagonists on feeding induced by peripheral, and raphe injection of 8-OH-DPAT in free-feeding rats. Following SC injection naloxone (0.1–10 mg/kg) dose-dependently reduced the feeding response induced by 100 μg/kg 8-OH-DPAT (SC). The lowest effective dose of naloxone was 1 mg/kg. This dose of naloxone also suppressed feeding induced by 8-OH-DPAT injected into either the dorsal (1 μg) or median (0.5 μg) raphe. Microinjecting 2 μg naloxone together with 8-OH-DPAT into either of these sites failed to prevent the increased feeding. These results indicate that the effects of naloxone are mediated at sites distal to the raphe nuclei. One possible site may be the nucleus accumbens, since methyl-naltrexone (0.3, 1 or 3 μg) injected into this site blocked the feeding responses to intra-raphe 8-OH-DPAT. Examination of the effects of naloxone and methyl-naltrexone in food-deprived rats ruled out intrinsic anorectic actions of these drugs as an explanation for the antagonism of 8-OH-DPAT-induced feeding. The results may indicate that reduced 5-HT function increases opiate activity in the nucleus accumbens. Alternatively opiate antagonists may prevent the indirect facilitation of dopamine activity resulting from a reduction in 5-HT neurotransmission. Opiate and dopamine systems in the nucleus accumbens play a major role in reward processes. Since opiate and dopamine antagonists reverse 8-OH-DPAT-induced feeding the possible role of reward mechanisms in mediating the effect of 8-OH-DPAT is discussed.     

Modulation of brain and pituitary dopamine receptors by estrogens and prolactin

1. Ovariectomized rats were treated for 2 weeks with 17β-estradiol (0.0002–100 μg/day). [³H] spiperone striatal dopamine receptor binding was maximally increased by 30% after 0.05 μg/day of 17β-estradiol and a similar increase is observed at higher doses. By contrast, plasma prolactin concentrations of these rats are unchanged after 0.05 μg/day and increased after 100 μg/day. A chronic estradiol treatment at very low doses (0.0002–0.001 μg/day) leads to increases in pituitary dopamine receptor binding while plasma prolactin levels are unchanged. At higher doses (1–100 μg/day) binding is decreased and plasma prolactin concentrations are elevated.

2. [³H]spiperone striatal dopamine receptor binding is elevated in lactating female rats compared to intact or ovariectomized female rats.

3. Anterior pituitary dopamine receptor concentrations fluctuate during the estrous cycle while striatal dopamine receptors are unchanged.

4. An injection of 30 ng of estradiol, which reproduces the estradiol proestrus surge, leads as in proestrus, to a decrease of anterior pituitary dopamine receptors.     

Blockade of hippocampal M1 muscarinic receptors impairs working memory performance of rats

In order to clarify the roles of hippocampal M₁ and M₂ muscarinic receptors in working and reference memory performance of rats, the effects of intrahippocampal injections of selective antagonists at both receptors on this behavior were examined with a three-panel runway task. In the working memory task, the M₁ muscarinic receptor antagonist pirenzepine, injected bilaterally at 0.32 and 1.0 μg/side into the dorsal hippocampus, significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). This effect of intrahippocampal pirenzepine (1.0 μg/side) on working memory was attenuated by concurrent injection of 10 μg/side AF102B, the selective M₁ muscarinic receptor agonist. Intrahippocampal injection of the M₂ muscarinic receptor antagonist methoctramine at doses up to 1.0 μg/side had no significant effect on the number of working memory errors. Intrahippocampal methoctramine injection at 3.2 μg/side produced a significant increase in working memory errors, an effect that was reversed by concurrent injection of 10 μg/side AF102B. Concurrent injection of 0.32 μg/side methoctramine significantly reduced the increase in working memory errors induced by intrahippocampal pirenzepine (1.0 μg/side). In the reference memory task, neither pirenzepine nor methoctramine affected the number of errors when injected into the hippocampus at doses up to 1.0 and 3.2 μg/side, respectively. These results suggest that processes mediated by M₁ muscarinic receptors in the hippocampus are involved in working memory, but not in reference memory, and that blockade of hippocampal M₂ muscarinic receptors ameliorates working memory deficits produced by M₁ muscarinic blockade, possibly by increasing acetylcholine release.     

5-HT receptors and hyper- or hypothermia: Elucidation by catecholamine antagonists injected into the cat hypothalamus

The receptor mechanisms in the hypothalamus and preoptic area which mediate thermoregulatory changes produced by serotonin (5-HT) were examined in the unrestrained cat. Stainless steel guide tubes were implanted just above the rostral diencephalon or preoptic area in each of twelve cats. 5-HT in a dose of 1.5 to 10.5 μg was micro-injected in a volume of 0.75 to 1.5 μl into a total of 256 test sites in the hypothalamus. An individual site at which 5-HT evoked a rise or a fall in core temperature of 0.5°C within 30 min was considered to be reactive to the indoleamine. Within such a region of maximum sensitivity, i.e., the cat's anterior hypothalamic, preoptic area (AH/POA), either norepinephrine (NE) or dopamine (DA) micro-injected in a dose of 2.33 to 14.0 μg in similar volumes evoked only a dose-dependent decline in the body temperature of the cat. 5-HT was found to cause either hyper- or hypothermia. The direction of temperature change caused by 5-HT was dependent principally upon the neuroanatomical locus of injection. Each of the 5-HT reactive sites was characterized pharmacologically by their pre-treatment with phentolamine (PHT), d-butaclamol (BUTAC) or methysergide (METHY) in doses of 1.0 to 10.0 μg. At the most rostral sites in POA, the catecholamine receptor antagonists selectively delayed the onset of the 5-HT hypothermia and limited the magnitude of the response. Within sites located in the AH in which 5-HT induced only a rise in body temperature, prior treatment of the site with the catecholamine receptor antagonists PHT or BUTAC failed to modify the response. These results indicate that 5-HT in producing hypothermia not only may saturate the 5-HT receptor sites but also may be taken up by catecholamine receptors which mediate the diencephalic heat loss pathway.