Evidence for a complex influence of nicotinic acetylcholine receptors on hippocampal serotonin release

The effects of nicotine on 5-hydroxytryptamine (5-HT) release from serotonergic nerve endings in rat dorsal hippocampal slices were studied. Nicotine (50-500 microM:) caused a concentration-dependent increase in 5-HT release. This effect was antagonised by mecamylamine (0.5 microM:), indicating an action at nicotinic receptors. Nicotine-evoked 5-HT release was not affected by tetrodotoxin (3 microM:), cadmium chloride (0.1 mM:), or the absence of Ca(2+) or Na(+) in the superfusion medium. Unexpectedly, higher concentrations of mecamylamine alone (1-50 microM:) increased 5-HT release. This suggested the presence of inhibitory input to 5-HT neurones and that these inhibitory neurones possess tonically active nicotinic receptors. The effect of mecamylamine (50 microM:) on 5-HT release was reduced by the muscarinic M(1) receptor agonist, McN-A-343 (100 microM:), but pirenzepine (0.005-1 microM:), which blocks M(1) receptors, alone increased 5-HT release. Hippocampal serotonergic neurones are known to possess both excitatory nicotinic receptors and inhibitory M(1) receptors. Although there may be several explanations for our results, one possible explanation is that nicotine stimulates 5-HT release by activating nicotinic heteroreceptors on 5-HT terminals. Mecamylamine (0.5 microM:) antagonises this effect, but higher concentrations increase 5-HT release indirectly by blocking the action of endogenous acetylcholine on nicotinic receptors situated on cholinergic neurones that provide muscarinic inhibitory input to 5-HT neurones.     

Increased hemorrhagic transformation and altered infarct size and localization after experimental stroke in a rat model type 2 diabetes

Background  

Interruption of flow through of cerebral blood vessels results in acute ischemic stroke. Subsequent breakdown of the blood brain barrier increases cerebral injury by the development of vasogenic edema and secondary hemorrhage known as hemorrhagic transformation (HT). Diabetes is a risk factor for stroke as well as poor outcome of stroke. The current study tested the hypothesis that diabetes-induced changes in the cerebral vasculature increase the risk of HT and augment ischemic injury.     

Correlational analysis for identifying genes whose regulation contributes to chronic neuropathic pain

Background

Descending facilitation, from the brainstem, promotes spinal neuronal hyperexcitability and behavioural hypersensitivity in many chronic pain states. We have previously demonstrated enhanced descending facilitation onto dorsal horn neurones in a neuropathic pain model, and shown this to enable the analgesic effectiveness of gabapentin. Here we have tested if this hypothesis applies to other pain states by using a combination of approaches in a rat model of osteoarthritis (OA) to ascertain if 1) a role for descending 5HT mediated facilitation exists, and 2) if pregabalin (a newer analogue of gabapentin) is an effective antinociceptive agent in this model. Further, quantitative-PCR experiments were undertaken to analyse the α₂δ-1 and 5-HT3A subunit mRNA levels in L3–6 DRG in order to assess whether changes in these molecular substrates have a bearing on the pharmacological effects of ondansetron and pregabalin in OA.

Results

Osteoarthritis was induced via intra-articular injection of monosodium iodoacetate (MIA) into the knee joint. Control animals were injected with 0.9% saline. Two weeks later in vivo electrophysiology was performed, comparing the effects of spinal ondansetron (10–100 μg/50 μl) or systemic pregabalin (0.3 – 10 mg/kg) on evoked responses of dorsal horn neurones to electrical, mechanical and thermal stimuli in MIA or control rats. In MIA rats, ondansetron significantly inhibited the evoked responses to both innocuous and noxious natural evoked neuronal responses, whereas only inhibition of noxious evoked responses was seen in controls. Pregabalin significantly inhibited neuronal responses in the MIA rats only; this effect was blocked by a pre-administration of spinal ondansetron. Analysis of α₂δ-1 and 5-HT3A subunit mRNA levels in L3–6 DRG revealed a significant increase in α₂δ-1 levels in ipsilateral L3&4 DRG in MIA rats. 5-HT3A subunit mRNA levels were unchanged.

Conclusion

These data suggest descending serotonergic facilitation plays a role in mediating the brush and innocuous mechanical punctate evoked neuronal responses in MIA rats, suggesting an adaptive change in the excitatory serotonergic drive modulating low threshold evoked neuronal responses in MIA-induced OA pain. This alteration in excitatory serotonergic drive, alongside an increase in α₂δ-1 mRNA levels, may underlie pregabalin's state dependent effects in this model of chronic pain.     

5-Hydroxytryptamine binding to synaptic membranes from rat brain.

The ³H-5HT binding capacity of rat brain synaptic membranes prepared by density gradient centrifugation has been investigated using a rapid ultrafiltration technique. A saturable, high affinity (K_D = 1.10^?9 M), 5HT displaceable binding has been found. It is thermosensitive, temperature dependent and pH dependent. 5HT and related tryptamines are the most effective displacers of bound ³H-5HT, whereas compounds which are not structurally related to 5HT (chlorpromazine, imipramine, cyproheptadine and cinanserine) and other neuro-transmitters (noradrenalin, dopamine) are ineffective. The distribution of 5HT-specific binding sites in the brain is related to serotonergic input. We conclude that these 5HT binding sites might possibly represent 5HT receptor sites.     

Chronic exercise improves repeated restraint stress-induced anxiety and depression through 5HT1A receptor and cAMP signaling in hippocampus

[Purpose]

Mood disorders such as anxiety and depression are prevalent psychiatric illness, but the role of 5HT1A in the anti-depressive effects of exercise has been rarely known yet. We investigated whether long-term exercise affected a depressive-like behavior and a hippocampal 5HT1A receptor-mediated cAMP/PKA/CREB signaling in depression mice model.

[Methods]

To induce depressive behaviors, mice were subjected to 14 consecutive days of restraint stress (2 hours/day). Depression-like behaviors were measured by forced swimming test (TST), and anxiety-like behavior was assessed by elevated plus maze (EPM). Treadmill exercise was performed with 19 m/min for 60 min/day, 5 days/week from weeks 0 to 8. Restraint stress was started at week 6 week and ended at week 8. To elucidate the role of 5HT1A in depression, the immunoreactivities of 5HT1A were detected in hippocampus using immunohistochemical technique.

[Results]

Chronic/repeated restraint stress induced behavioral anxiety and depression, such as reduced time and entries in open arms in EPM and enhanced immobility time in FST. These anxiety and depressive behaviors were ameliorated by chronic exercise. Also, these behavioral changes were concurrent with the deficit of 5HT1A and cAMP/PKA/CREB cascade in hippocampus, which was coped with chronic exercise.

[Conclusion]

These results suggest that chronic exercise may improve the disturbance of hippocampal 5HT1A-regulated cAMP/PKA/CREB signaling in a depressed brain, thereby exerting an antidepressive action.     

Flux coupling in the human serotonin transporter

The serotonin (5-hydroxytryptamine; 5HT) transporter (SERT) catalyzes the movement of 5HT across cellular membranes. In the brain, SERT clears 5HT from extracellular spaces, modulating the strength and duration of serotonergic signaling. SERT is also an important pharmacological target for antidepressants and drugs of abuse. We have studied the flux of radio-labeled 5HT through the transporter stably expressed in HEK-293 cells. Analysis of the time course of net transport, the equilibrium 5HT gradient sustained, and the ratio of the unidirectional influx to efflux of 5HT indicate that mechanistically, human SERT functions as a 5HT channel rather than a classical carrier. This is especially apparent at relatively high [5HT](out) (> or =10 microM), but is not restricted to this regime of external 5HT.     

Human umbilical cord mesenchymal stromal cells as an adjunct therapy with therapeutic hypothermia in a piglet model of perinatal asphyxia

Background: With therapeutic hypothermia (HT) for neonatal encephalopathy, disability rates are reduced, but not all babies benefit. Pre-clinical rodent studies suggest mesenchymal stromal cells (MSCs) augment HT protection. Aims:The authors studied the efficacy of intravenous (IV) or intranasal (IN) human umbilical cord-derived MSCs (huMSCs) as adjunct therapy to HT in a piglet model. Methods:A total of 17 newborn piglets underwent transient cerebral hypoxia-ischemia (HI) and were then randomized to (i) HT at 33.5°C 1–13 h after HI (n = 7), (ii) HT+IV huMSCs (30 × 10⁶ cells) at 24 h and 48 h after HI (n = 5) or (iii) HT+IN huMSCs (30 × 10⁶ cells) at 24 h and 48 h after HI (n = 5). Phosphorus-31 and hydrogen-1 magnetic resonance spectroscopy (MRS) was performed at 30 h and 72 h and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and oligodendrocytes quantified. In two further piglets, 30 × 10⁶ IN PKH-labeled huMSCs were administered. Results:HI severity was similar between groups. Amplitude-integrated electroencephalogram (aEEG) recovery was more rapid for HT+IN huMSCs compared with HT from 25 h to 42 h and 49 h to 54 h (P ≤ 0.05). MRS phosphocreatine/inorganic phosphate was higher on day 2 in HT+IN huMSCs than HT (P = 0.035). Comparing HT+IN huMSCs with HT and HT+IV huMSCs, there were increased OLIG2 counts in hippocampus (P = 0.011 and 0.018, respectively), internal capsule (P = 0.013 and 0.037, respectively) and periventricular white matter (P = 0.15 for IN versus IV huMSCs). Reduced TUNEL-positive cells were seen in internal capsule with HT+IN huMSCs versus HT (P = 0.05). PKH-labeled huMSCs were detected in the brain 12 h after IN administration. Conclusions:After global HI, compared with HT alone, the authors saw beneficial effects of HT+IN huMSCs administered at 24 h and 48 h (30 × 10⁶ cells/kg total dose) based on more rapid aEEG recovery, improved ³¹P MRS brain energy metabolism and increased oligodendrocyte survival at 72 h.     

mCPP but not TFMPP is an antagonist at cardiac 5HT3 receptors.

The prototypic arylpiperazines, meta-chlorophenylpiperazine (mCPP), meta-trifluoromethylphenylpiperazine (TFMPP) and quipazine are widely studied serotonergic ligands with nonselective effects at 5HT1 and 5HT2 receptor subtypes. The present study was designed to compare the affinities of these arylipiperazines at 5HT3 receptors, and to determine agonist or antagonist activity at 5HT3 receptors. Quipazine showed high affinity at brain 5HT3 receptors (IC50 = 4.4 nM) and was a potent agonist of the von Bezold-Jarisch reflex in anesthetized rats, a response mediated by cardiac 5HT3 receptors. In concentrations that activated 5HT3 receptors, quipazine also antagonized serotonin-induced bradycardia in anesthetized rats. Taken together, these data suggest that quipazine is an agonist/antagonist with high affinity at 5HT3 receptors in both brain and cardiac tissue. Although mCPP also showed relatively high affinity at brain 5HT3 receptors (IC50 = 61.4 nM), it did not activate the von Bezold-Jarisch reflex; instead, mCPP potently antagonized serotonin-induced bradycardia. Thus, mCPP acts as an antagonist at 5HT3 receptors in the periphery. Although both quipazine and mCPP possessed relatively high affinity at brain 5HT3 receptors, TFMPP did not bind appreciably to 5HT3 receptors in brain (IC50 = 2373 nM) and neither activated nor inhibited cardiac 5HT3 receptors. That TFMPP did not interact with 5HT3 receptors, whereas quipazine and mCPP did, is in marked contrast to the similar effects of all three arylpiperazines at other serotonin receptors. The selectivity of TFMPP for 5HT1 and 5HT2 receptors (i.e., its minimal affinity for 5HT3 receptors) suggests that this arylpiperazine may be a preferred ligand relative to mCPP when studying 5HT1 or 5HT2 receptor mediated responses.     

Striatal serotonin 2C receptors decrease nigrostriatal dopamine release by increasing GABA‐A receptor tone in the substantia nigra

Drugs acting at the serotonin‐2C (5‐HT2C) receptor subtype have shown promise as therapeutics in multiple syndromes including obesity, depression, and Parkinson's disease. While it is established that 5‐HT2C receptor stimulation inhibits DA release, the neural circuits and the localization of the relevant 5‐HT2C receptors remain unknown. This study used dual‐probe in vivo microdialysis to investigate the relative contributions of 5‐HT2C receptors localized in the rat substantia nigra (SN) and caudate‐putamen (CP) in the control of nigrostriatal DA release. Systemic administration (3.0 mg/kg) of the 5‐HT2C receptor selective agonist Ro 60‐0175 [(αS)‐6‐Chloro‐5‐fluoro‐α‐methyl‐1H‐indole‐1‐ethanamine fumarate] decreased, whereas intrastriatal infusions of the selective 5‐HT2C antagonist SB 242084 [6‐Chloro‐2,3‐dihydro‐5‐methyl‐N‐[6‐[(2‐methyl‐3‐pyridinyl)oxy]‐3‐pyridinyl]‐1H‐indole‐1‐carboxyamide; 1.0 μM] increased, basal DA in the CP. Depending on the site within the SN pars reticulata (SNpr), infusions of SB 242084 had more modest but significant effects. Moreover, infusions of the GABA‐A receptor agonist muscimol (10 μM) into the SNpr completely reversed the increases in striatal DA release produced by intrastriatal infusions of SB 242084. These findings suggest a role for 5‐HT2C receptors regulating striatal DA release that is highly localized. 5‐HT2C receptors localized in the striatum may represent a primary site of action that is mediated by the actions on GABAergic activity in the SN.

     

Association of [3H]-Imipramine and [3H]-Paroxetine Binding with the 5Ht Transporter in Brain and Platelets Relevance to Studies in Depression

Abstract

[³H]-Imipramine and [³H]-paroxetine label with high affinity a site which is associated with the serotonergic transporter in brain and platelets. The pharmacological profile of inhibition by drugs of [³H]-imipramine and [³H]-paroxetine binding is highly correlated with the potency of the drugs to inhibit the uptake of 5HT.

Denervation of serotonergic neurons by electrolytic lesions or with 5,7-dihydroxytryptamine produces marked decreases in the density of [³H]-imipramine as well as [³H]-paroxetine binding. Dissociation kinetic experiments support the view that the substrate recognition site for 5HT is different from the modulatory site which is labelled by [³H]-imipramine or [³H]-paroxetine. The existence of an endogenous ligand acting on the [³H]-imipramine recognition site to modulate the 5HT transporter was proposed by several laboratories.     

Serotonergic activation rescues reproductive function in fasted mice: does serotonin mediate the metabolic effects of leptin on reproduction?

Negative energy balance inhibits reproduction by restraining GnRH secretion. Leptin is a permissive metabolic signal for reproduction, but GnRH neurons do not appear to express leptin receptors, suggesting that interneurons transmit leptin signals to these cells. Serotonin (5HT) has satiety effects similar to those of leptin and alters LH release, and serotonergic neurons, which have been shown to express leptin receptors, terminate on GnRH neurons. We hypothesized that serotonergic neurons convey leptin signals to the reproductive neuroendocrine axis. To test this, mice were fasted for 48 h beginning on Diestrous Day 1. While fasting, mice received saline or leptin every 12 h or the 5HT-selective reuptake-inhibitor fluoxetine once at the start of the fast. Estrous cycles of fasted mice were longer (mean +/- SEM, 10.2 +/- 0.5 days; P < 0.0001) than those of fed mice (4.5 +/- 0.2 days). As previously reported, leptin prevented fasting-induced cycle lengthening (4.6 +/- 0.7 days). Fluoxetine also rescued estrous cycles in fasted mice (4.7 +/- 0.6 days), suggesting that 5HT and leptin have similar positive effects on reproduction. Coadministration of the 5HT 1/2/7 receptor-antagonist metergoline blocked rescue of cycle length by fluoxetine and by leptin. Treating leptin-deficient ob/ob and leptin receptor-deficient db/db mice with fluoxetine did not normalize body weight or rescue fertility, perhaps due to altered serotonergic tone in these animals. Together, these data demonstrate a permissive role for serotonergic systems in the metabolic control of reproduction and are consistent with the hypothesis that serotonergic neurons convey leptin signals to GnRH neurons.     

Multiple mechanisms of serotonergic signal transduction

In this article we review serotonergic signal transduction mechanisms in the central and peripheral nervous systems and in a variety of target organs. The various classes of pharmacologically defined serotonergic receptors are coupled to three major effector systems: (1) adenylate cyclase; (2) phospholipase C mediated phosphoinositide (PI) hydrolysis and (3) ion channels (K+ and Ca++). Long term occupancy of serotonergic receptors also appears to induce alterations in mRNA and protein synthesis. For all three types of signal transduction there is evidence accumulating which suggests the involvement of guanine nucleotide regulatory proteins. Recent findings suggest that the distinct types of pharmacologically defined serotonergic receptors (5HT1A, 5HT1B, 5HT1c, 5HT2) may be coupled to one or more signal transduction systems. Thus, 5HT1 receptors may both activate and inhibit adenylate cyclase and increase K+-ion conductance in the hippocampus. 5HT2 receptors which activate PI hydrolysis in the brain, both open voltage-gated calcium channels and activate PI metabolism in certain smooth muscle preparations. Thus, each class of serotonergic receptor may be linked to one or more distinct biochemical transduction mechanisms. The possibility is raised that selective agonists and antagonists might be developed which have specific effects on a particular receptor-linked effector system.     

Prospective study on dosimetric comparison of helical tomotherapy and 3DCRT for craniospinal irradiation – A single institution experience

Aim

This prospective study aims to assess feasibility of helical tomotherapy (HT) for craniospinal irradiation (CSI) and perform dosimetric comparison of treatment plans for both HT and 3D conformal radiotherapy (3DCRT).

Background

CSI is a challenging procedure. Large PTV size requires field matching due to technical limitations of standard linear accelerators, which cannot irradiate such volumes as a single field. HT could help to avoid these limitations as irradiation of long fields is possible without field matching.

Materials and methods

Three adults were enrolled from 2009 to 2010. All patients received radiochemotherapy. Treatment plans in prone position for 3DCRT and in supine position for HT were generated. The superior plan was used for patients’ irradiation. Plans were compared with the application of DVH, Dx parameters – where x represents a percentage of the structure volume receiving a normalized dose and homogeneity index (HI).

Results

All patients received HT irradiation. The treatment was well tolerated. The HT plans resulted in a better dose coverage and uniformity in the PTV: HI were 5.4, 7.8, 6.8 for HT vs. 10.3, 6.6, 10.4 for 3DCRT. For most organs at risk (OARs), the D(V80) was higher for HT than for 3DCRT, whereas D(V5) was lower for HT.

Conclusions

HT is feasible for CSI, and in comparison with 3DCRT it improves PTV coverage. HT reduces high dose volumes of OARs, but larger volumes of normal tissue receive low radiation dose. HT requires further study to establish correlations between dosimetrical findings and clinical outcomes, especially with regard to late sequelae of treatment.     

The affinity of metergoline for 3H-Serotonin (5HT) binding sites is regulated by guanine nucleotide

100 μM guanine nucleotide Gpp (NH)p reduces the affinity of the serotonergic antagonist metergoline for ³H-5HT binding sites in rat cerebral cortex. This effect is present both in inhibition binding and in saturation experiments. The hypothesis that the interaction of some serotonergic antagonists with ³H-5HT binding sites is regulated by guanine nucleotides is discussed.     

Selective in vivo labelling of serotonergic neurones by 5,6-dihydroxytryptamine in the snail Helix pomatia L

1. In the central nervous system of Helix pomatia L. serotonergic neurones were labelled in vivo by injecting 5,6-dihydroxytryptamine into the body cavity of the animals. By the 60th day after the injection, all serotonergie neurones contained a strong, rusty-brown pigmentation located to large lysosomes in the cells.2. The membrane potential, action and synaptic potential generating abilities of the pigmented serotonergic neurones were normal. Their input relations from the cardio-renal system and the lip, respectively, were as intact as in the untreated animals.3. The opportunity to study in vivo labelled and normally functioning neurones may contribute to the better understanding of their function.     

Traumatic brain injury and the effects of diazepam,diltiazem, and MK-801 on GABA-A receptor subunit expression in rat hippocampus

Background  

Excitatory amino acid release and subsequent biochemical cascades following traumatic brain injury (TBI) have been well documented, especially glutamate-related excitotoxicity. The effects of TBI on the essential functions of inhibitory GABA-A receptors, however, are poorly understood.     

Long-term tryptophan administration enhances cognitive performance and increases 5HT metabolism in the hippocampus of female rats

Summary. It has been shown in various studies that increase in serotonergic neurotransmission is associated with increased memory consolidation whereas low brain 5HT impairs memory performance. In the first phase of our study we found that tryptophan (TRP) administration for 6 weeks increased plasma TRP and whole brain TRP, 5HT and 5HIAA levels. Many brain regions are involved in the learning process but particularly the hippocampus is known to have key role in learning and memory. The present study was therefore designed to investigate the effects of TRP loading particularly on hippocampal 5HT metabolism and cognitive performance in rats. TRP-treated rats demonstrated spatial enhancement as evidenced by a significant decrease in time to find the hidden food reward in radial arm maze test (RAM). The important finding of the present study was the greater increase in the 5HT metabolism in hippocampus than in any other brain region of the TRP-treated rats. This increased 5HT metabolism in the hippocampus emphasizes the involvement of this region in memory process.     

Twenty-four hour rhythms in serum and brain indoleamine concentrations: tryptophan-5-hydroxylase and monoamine oxidase activity in the rat.

The relationship between the 24 h rhythm in 5-hydroxy-tryptamine (5HT) levels in rat brain, the availability of precursors of 5HT and the concentration of its major metabolite, 5-hydroxyindole acetic acid (5HIAA) has been investigated. Serum total and "free" tryptophan (TRY) levels and brain TRY levels all show a 24 h rhythm with highest concentrations in the middle of the dark phase i.e. 12 h displaced from that of the 5HT rhythm. No 24 h variation in either tryptophan-5-hydroxylase or monoamine oxidase activity was detected, nor did brain 5-hydroxytryptophan (5HTP) levels vary with clock hour. Changes in 5HIAA concentration paralleled those of 5Ht. The uptake of 14C-5HTP, 14C-TRY and 14C-5HT into homogenates of the septal region of rat brain did not display a circadian rhythm, although there was evidence that uptake of 14C-TRY in an isolated synaptosomal preparation from the same region was greater during the light phase, indicating the possibility that uptake of the precursor into the nerve ending may be, in part, responsible for the 24 h rhythm in brain 5HT. It is concluded that brain 5HT levels are independent of the serum or brain TRY concentrations measured. Since changes in 5HT with clock hour are paralleled by changes in 5HIAA, it also seems unlikely that the increase in brain 5HT during the light phase is caused by a decreased release of 5HT from nerve endings.