Urethane-anaesthetized Rats Research Articles

Involvement of central β2-adrenergic, NMDA and thromboxane A2 receptors in the pressor effect of anandamide in rats

Intravenous (i.v.) injection of the endocannabinoid anandamide induces triphasic cardiovascular responses, including a pressor effect mediated via unknown central and peripheral mechanism(s). The aim of the present study was to determine the central mechanism(s) responsible for the pressor response to anandamide. For this purpose, the influence of antagonists at thromboxane A(2) TP (sulotroban, daltroban, SQ 29548), NMDA (MK-801) and beta(2)-adrenergic receptors (ICI 118551) on the pressor effect induced by i.v. and intracerebroventricularly (i.c.v.) administered anandamide was examined in urethane-anaesthetized rats. Anandamide (1.5-3 micromol/kg, i.v.) or its stable analogue methanandamide (0.75 micromol/kg, i.v.) increased blood pressure by 25%. Anandamide (0.03 mumol per animal i.c.v.) caused a pure pressor effect (by 20%) but only in the presence of antagonists of CB(1) and TRPV1 receptors. The effects of cannabinoids (i.v. or i.c.v.) were diminished by i.v. daltroban, sulotroban (10 mumol/kg each), and/or SQ 29548 (1 mumol/kg). The effect of anandamide i.v. was reduced by SQ 29548 (0.02 mumol per animal i.c.v.) and by the thromboxane A(2) synthesis inhibitor furegrelate i.c.v. (1.8 micromol per animal). ICI 118551, MK-801 (1 micromol/kg i.v. each), and bilateral adrenalectomy diminished the effect of anandamide i.c.v. Sulotroban (i.v.) failed to affect the response to anandamide (i.v.) in pithed rats, and anandamide and methanandamide did not bind to TP receptors in rat platelets. The present study suggests that central beta(2)-adrenergic, NMDA and thromboxane A(2) receptors are involved in the anandamide-induced adrenal secretion of catecholamines and their pressor effect in urethane-anaesthetized rats.

Responses to cholecystokinin in the ventromedial nucleus of the rat hypothalamus in vivo

The peptide cholecystokinin (CCK) is a short-term satiety signal released from the gastrointestinal tract during food intake. From the periphery, CCK signalling travels via the vagus nerve to reach the brainstem from which it is relayed higher into the brain. The hypothalamus is a key integrator of appetite-related stimuli and the ventromedial nucleus of the hypothalamus (VMN) is thought to have an important role in the regulation of satiety. We investigated the effect of intravenous injections of CCK on the spontaneous firing activity of single VMN neurons in urethane-anaesthetised rats in vivo. We found that the predominant effect of CCK on the electrical activity in the VMN is inhibitory. We analysed the responses to CCK according to electrophysiologically distinct subpopulations of VMN neurons and found that four of these VMN subpopulations were inhibited by CCK, while five were not significantly affected. Finally, CCK-induced inhibitory response in VMN neurons was not altered by pre-administration of intravenous leptin.

Influence of dietary sodium on the blood pressure and renal sympathetic nerve activity responses to intracerebroventricular angiotensin II and angiotensin III in anaesthetized rats

The regulation of blood pressure and sympathetic outflow by the brain renin-angiotensin system in animals subjected to raised or lowered dietary Na(+) intake is unclear. This study compared the mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) responses to intracerebroventricular (i.c.v.) infusion of angiotensin II (AngII) and III (AngIII) before and after peripheral V(1) receptor blockade (V(1)B) in alpha-chloralose-urethane-anaesthetized rats fed a low (0.03%, LNa(+)), normal (0.3%, NNa(+)) or high Na(+) diet (3.0%, HNa(+)) from 4 to 11 weeks of age. The rise in MAP 2 min post AngII i.c.v. was greater in HNa(+) (14 +/- 3 mmHg) versus LNa(+) (8 +/- 1 mmHg, P < 0.05) and after AngIII i.c.v. in HNa(+) (14 +/- 3 mmHg) versus NNa(+) (6 +/- 1 mmHg, P < 0.05) and LNa(+) (7 +/- 1 mmHg, P < 0.05). The MAP responses to AngII and AngIII i.c.v. were abolished after V(1)B in LNa(+), but were only attenuated in HNa(+). In NNa(+), V(1)B blunted the MAP responses to AngII and abolished those to AngIII. The MAP remained elevated 30 min after AngII in all groups, but returned to baseline levels 15 min after AngIII in NNa(+) and HNa(+) (P < 0.01). Twenty minutes after i.c.v. AngII, RSNA rose above baseline in HNa(+) (112 +/- 1%), a response not observed in the LNa(+) and NNa(+) groups. Twenty minutes post AngIII i.c.v., RSNA was elevated in both HNa (109 +/- 2%) and NNa(+) (109 +/- 2%). After V(1)B, RSNA rose only in the HNa(+) group 15 min post AngIII infusion (109 +/- 1%). Together, these findings: (1) suggest that HNa(+) intake augments the MAP and RSNA responses to i.c.v. AngII and AngIII; (2) highlight an important role for peripheral V(1) receptors during these responses; and (3) differentiate the effects of AngII and AngIII on blood pressure and RSNA.

Nitric oxide modulates the discharge rate of basal forebrain neurones: a study in freely moving rats

In urethane-anaesthetized rats the infusion of a nitric oxide (NO)-donor [NOC-18, 1 mM (DETA/NO); 2,2'-(hydroxynitrosohydrazino)bis-ethanamine)] into the basal forebrain (BF) inhibited the discharge rate of most neurones, suggesting that NO may promote sleep via inhibition of wake-promoting neurones in the BF. However, this hypothesis still needs to be confirmed in freely moving rats. The objective of this study was to examine whether NO modulates the discharge rate of BF neurones in freely moving rats in a similar manner to anaesthetized rats. We measured the discharge rates of BF neurones in freely moving rats during microdialysis infusion of a NO-donor (1 mm; NOC-18) in different vigilance states. Neurones were characterized as wake (W)-on (51.8%), W-off (28.6%) and W/non-rapid eye movement (REM)-independent (21.4%) based on their discharge profiles during wakefulness (W) and non-REM sleep. The NO-donor affected the discharge rate of most BF neurones during quiet wakefulness (QW; 55%) and non-REM sleep (64%). The most prominent response in all neuronal groups was a decrease in the discharge rate during QW and non-REM sleep. A small subpopulation of neurones increased the discharge rate. The increase in NO in the BF during prolonged wakefulness may facilitate sleep via inhibition of wake-promoting neurones.

Daily Rhythms of Spike Coding in the Rat Supraoptic Nucleus

Novel measures of coding based on interspike intervals were used to characterise the rhythms of single unit activity in the supraoptic nucleus during the day/night cycle in urethane-anaesthetised rats in vivo. Both continuously firing and phasic cells showed significant (P < 0.001) diurnal rhythms of spike frequency and in the irregularity of firing, as quantified by the log interval entropy (ENT). Comparison of rhythms in log interval ENT showed that the amplitude of the rhythms was greater for the continuously firing cells than for the phasic cells (P = 0.002). Rhythms persisted after hypertonic stimulation or pinealectomy and both treatments reduced the amplitude significantly only for the continuously firing cell group. By contrast, the mesor (i.e. mid-point of the rhythm) was reduced only for the phasic cell group. A similar analysis applied to the activity of cells of the suprachiasmatic nucleus showed that, after pinealectomy, there was a significant rhythm in ENT (P < 0.001) but not firing rate; however, the amplitude of the rhythm in ENT was attenuated (P = 0.047). Diurnal changes in the electrical activity of supraoptic cells are consistent with previously reported circadian changes in magnocellular neuropeptide release. Differences between continuous and phasic cell groups in the effects of osmotic stimulation on rhythmic activity indicate that the two cell types differ in their coding of osmolality and zeitgeber time information. The different effects of pinealectomy on the supraoptic and suprachiasmatic nuclei suggest that removal of endogenous melatonin unmasks a difference in circadian coding between the two nuclei.

Muscarinic receptor activation in the lumbosacral spinal cord ameliorates bladder irritation in rat cystitis models

To investigate whether activation of spinal cholinergic pathways affects bladder activity in rats with chemical cystitis induced by acetic acid (AA) and cyclophosphamide (CYP). The effects of intrathecal (i.t.) application of neostigmine as an acetylcholine esterase (AChE) inhibitor, oxotremorine-M (OXO-M) as a muscarinic ACh receptor (mAChR) agonist or epibatidine as a nicotinic AChR (nACHR) agonist with or without atropine as a mAChR antagonist or mecamylamine (MEC) as a nAChR antagonist at the level of L6-S1 spinal cord on C-fibre mediated bladder irritation were examined by using continuous-infusion cystometry (0.1 mL/min) in urethane-anaesthetized female Spraque-Dawley rats. Bladder irritation was induced by intravesical AA (0.25%) or pretreatment with intraperitoneal injection of CYP (150 mg/kg) 24 h before cystometry. In control rats during intravesical saline, the effects of above drugs on the bladder activity were also examined. The intercontraction intervals (ICI) in AA- and CYP-treated rats were significantly shorter than those in control rats. Neostigmine (i.t.) significantly increased ICI dose-dependently without changing maximum voiding pressure in all groups. The mean (sem) maximal percentage increases in ICI after i.t. neostigmine as compared with the pretreatment value in control, AA- and CYP-treated rats were 93.2 (18.5)%, 117.5 (20.2)% and 200.7 (19.6)%, respectively. The percentage increases of ICI in the CYP-treated group were significantly (P < 0.05) higher than those in the AA-treated or control groups. In all groups, pretreatment with atropine, but not MEC, almost completely antagonized the inhibitory effects of neostigmine. OXO-M produced almost the same effects as that of neostigmine in all groups. Conversely, i.t. epibatidine decreased the ICI in all groups and these excitatory effects were completely antagonized by pretreatment with MEC and significantly inhibited by pretreatment with MK-801(noncompetitive n-methyl-d-aspartate receptor antagonist). These results indicate that accumulation of ACh by AChE inhibition in the spinal cord can ameliorate frequent urination in chemical cystitis via mAChRs, but not nAChRs.

Effects of cholinesterase inhibition in supraspinal and spinal neural pathways on the micturition reflex in rats

To investigate whether activation of brain and spinal cholinergic pathways affects the micturition reflex in rats. The effects of intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration of neostigmine as a cholinesterase inhibitor and oxotremorine-M (OXO-M) as a muscarinic acetylcholine receptor (mAChRs) agonist, on the micturition reflex were evaluated by infusion cystometrography (CMG) in urethane-anaesthetized untreated rats or rats pretreated with capsaicin. Neostigmine injected i.c.v. increased bladder capacity (BC) and pressure threshold (PT) dose-dependently, with an increase in maximum voiding pressure (MVP) and a decrease in voiding efficiency (VE) at higher doses. Also, neostigmine injected i.t. increased the BC and PT dose-dependently without changing MVP or VE, and these effects were not apparent in capsaicin-pretreated rats. In both routes, atropine as an antagonist of mAChRs, but not mecamylamine as a nicotinic-AChR antagonist, almost completely antagonized the effects of neostigmine. The rank order of potencies of the antagonists for increasing effects of BC induced by 1 nmol of neostigmine was: pirenzepine (an M(1) mAChR antagonist) = atropine > 4-DAMP (an M(3) mAChR antagonist) " methoctramine (an M(2) mAChR antagonist) and tropicamide (an M(4) mAChR antagonist) via the i.c.v. route; and atropine > methoctramine > pirenzepine > tropicamide and 4-DAMP via the i.t. route, respectively. OXO-M injected via i.c.v. and i.t. had the same effects on BC, PT, MVP and VE as neostigmine by i.c.v. and i.t., respectively. These results indicate that activation of muscarinic cholinergic mechanisms by the cholinesterase inhibitor in the brain and spinal cord can inhibit the micturition reflex, mainly by affecting afferent pathways. These mAChR-induced inhibitory effects seem to be mediated through M(1)/M(3) receptor subtypes in the brain, while in the spinal cord, the M(1)/M(2) receptor subtypes might be involved in inhibitory effects, which are mediated via inhibition of mechanoceptive C-fibre afferent pathways.

Cardiovascular effects of intravenous administered 26RFa, a novel RFamide peptide ligand for GPR103, in anaesthetised rats

26RFa, a novel RFamide-related peptide, has been identified as the endogenous ligand for GPR103, and the biological functions of this neuropeptide have not been well investigated. In the present study, the cardiovascular effects of intravenous administration of rat/mouse 26RFa were tested in rats. Intravenous administered 26RFa (100–800 nmol/kg, i.v.) caused a biphasic change in blood pressure, and an increase in heart rate in urethane-anaesthetised rats. The pressor effects induced by 26RFa were significantly inhibited by pretreatment with the α- and β-adrenoreceptor antagonists. In contrast, the tachycardiac responses to 26RFa were significantly attenuated by bilateral cervical vagotomy and β-adrenoreceptor antagonist propranolol. These data imply that the peripheral cardiovascular regulation of 26RFa might be involved in vagal components and catecholaminergic pathway. Furthermore, in order to evaluate the importance of the regions of 26RFa molecule in its cardiovascular regulation, the two C-terminal fragments of rat/mouse 26RFa, 26RFa(8–26) and 26RFa(19–26), were synthesized and investigated to address their peripheral cardiovascular responses in rats. Surprisingly, intravenous injections of 26RFa(8–26) and 26RFa(19–26) (50–300 nmol/kg, i.v.) produced dose-dependent increases in blood pressure and heart rate, which exerted different sensitivities to bilateral vagotomy and β-adrenergic receptor antagonist. The results indicate that intravenous administrations of 26RFa and its fragments induced their cardiovascular effects via different pathways, which further suggest that the N-terminal residues of 26RFa are required for its cardiovascular activities.

Potentiation of excitatory serotonergic responses by MK-801 in the medial prefrontal cortex

New atypical antipsychotics show a greater affinity to serotonergic rather than to dopamine receptors, suggesting that serotonin (5-HT) has a major role in the pathophysiology and treatment of schizophrenia. The goal of this study was to characterise the response of pyramidal neurons in the medial prefrontal cortex (mPFC) to 5-HT and NMDA before and after administration of the NMDA receptor antagonist, MK-801 (dizocilpine), a well-validated pharmacological model of psychosis. mPFC pyramidal (glutamatergic) neurons were recorded in urethane-anaesthetised rats. The responses to NMDA and 5-HT were assessed using in vivo electrophysiology and microiontophoresis. The 5-HT2A/2C antagonist ritanserin and the 5-HT1A antagonist WAY100635 were used to block 5-HT responses. MK-801 decreased the NMDA-induced excitatory responses and increased NMDA-evoked burst activity among mPFC pyramidal neurons. Three subpopulations of pyramidal cells were identified according to their responses to 5-HT: excitation (33%), inhibition (40%) and non-response (27%). The inhibitory responses were blocked by WAY100635 in 100% of cases, but not by ritanserin; the excitatory responses were blocked by ritanserin in 75% of cases, but not by WAY100635. The administration of MK-801 potentiated the firing rate of excitatory responses but did not modify the inhibitory responses induced by microiontophoretic application of 5-HT. These results suggest that MK-801 modifies 5-HT synapses in the mPFC by potentiating the excitatory 5-HT2A/2C responses and attenuating NMDA excitations. These data indicate that 5-HT excitatory transmission is selectively impaired at the mPFC level in this pharmacological model of schizophrenia.

Response of medial preoptic/anterior hypothalamic neurones to ventrolateral medulla stimulation and its attenuation by adrenoceptor antagonists.

Extracellular single-unit recordings were made from medial preoptic/anterior hypothalamic (MPOAH) neurones in urethane-anaesthetized female rats, and the responses of these neurones to electrical stimulation of the ventrolateral medulla (VLM) were investigated. Of 189 neurones tested, 20% were excited and 21% were inhibited following VLM stimulation. The latency of inhibitory responses showed a normal distribution and the mean latency was 47 ms, whereas the latency of excitatory responses showed a bimodal distribution with peaks in the ranges of 0-10 ms and 40-50 ms, respectively. Excitatory responses with latencies greater than 15 ms and inhibitory responses were significantly attenuated by intravenous injections of the alpha-adrenoceptor antagonist phenoxybenzamine but not the beta-adrenoceptor antagonist propranolol. In the light of these observations and the known anatomical and electrophysiological findings, it is suggested that A1 noradrenergic and/or C1 adrenergic inputs from the VLM to MPOAH neurones are, at least in part, mediated by alpha-adrenoceptors.

B2 kinin receptors mediate the Indian red scorpion venom‐induced augmentation of visceral reflexes via the nitric oxide cyclic guanosine monophosphate pathway

This study was performed to delineate the kinin (receptor)-dependent pathways in the Indian red scorpion (Mesobuthus tamulus; MBT) venom-induced pulmonary oedema as well as the augmentation of cardio-pulmonary reflexes evoked by phenyldiguanide (PDG). In urethane-anaesthetized adult rats, the effect of venom on the PDG reflex responses (blood pressure, heart rate and respiration rate) and the pulmonary water content was ascertained using various antagonists(des- Arg, B(1) receptor antagonist; Hoe 140, B(2) receptor antagonist; N(omega)-nitro-l-arginine methyl ester (l-NAME), nitric oxide (NO) synthase inhibitor; methylene blue, soluble guanylate cyclase inhibitor; and glibenclamide, K(+)(ATP) channel blocker). The effect of phosphodiesterase V inhibitor (sildenafil citrate) on the reflex response and the pulmonary water content was also examined and compared with venom-induced responses. Intravenous injection of PDG (10 microg kg(-1)) evoked apnoea, bradycardia and hypotension lasting >60 s. Exposure to MBT venom (100 microg kg(-1)) for 30 min augmented the PDG reflex responses by two times and increased the pulmonary water content, significantly. Hoe 140 blocked the venom-induced responses (augmentation of PDG reflex and increased pulmonary water content) whereas des-Arg did not. l-NAME, methylene blue or glibenclamide also blocked the venom-induced responses. Furthermore, sildenafil citrate (that increases cGMP levels) produced augmentation of PDG reflex response and increased the pulmonary water content as seen with venom. The results indicate that venom-induced responses involve B(2) kinin receptors via the NO-dependent guanylate cyclase-cGMP pathway involving K(+)(ATP) channels.

Complex cardiovascular actions of α‐adrenergic receptors expressed in the nucleus tractus solitarii of rats

Although both alpha(1)- and alpha(2)-adrenergic receptors (ARs) are known to be expressed in the nucleus of the solitary tract (NTS), the functional significance of these receptors is still not fully established. In this study, we microinjected alpha(1)- and alpha(2)-AR agonists into the NTS of urethane-anaesthetized Wister rats to study the cardiovascular effects in response to their activation. When the alpha(1)-AR agonist phenylephrine was microinjected into the area where barosensitive neurons are dominantly located (baro-NTS), mean arterial pressure (MAP) and heart rate (HR) were significantly elevated. When tested in the area where chemosensitive neurons are dominantly located (chemo-NTS), however, MAP and HR were significantly decreased. Pretreatment with the non-specific alpha-AR antagonist phentolamine into the NTS inhibited the phenylephrine-induced cardiovascular responses. In contrast, microinjection of the alpha(2)-AR agonist clonidine into either the baro-NTS or the chemo-NTS decreased MAP and HR; they were also inhibited by the alpha(2)-adrenergic antagonist yohimbine. Moreover, we immunohistochemically identified that cardiovascular responses induced by alpha(1)-ARs may be mediated by NTS neurons while those induced by alpha(2)-ARs may be mediated by astrocytes located in the barosensitive and chemosensitive areas of the NTS. These results suggest that both types of alpha-AR expressed in the NTS may be involved in regulating cardiovascular homeostasis via modulation of input signals from baroreceptor and chemoreceptor afferents; however, cardiovascular responses produced by stimulation of alpha(1)-ARs are strictly location specific within the NTS.

Effects of cervical vagus nerve stimulation on amygdala-evoked responses of the medial prefrontal cortex neurons in rat

In experiments on urethane-anaesthetized rats, the effects of repetitive vagus nerve stimulation (VNS) on responses of medial prefrontal cortex (mPFC) neurons to electrical stimulation of the basal nucleus of the amygdala were examined before and after intracerebroventricular administration of the neuronal nitric oxide synthase inhibitor 7-nitroindasole (7-NI). It was shown that the amygdala-evoked responses of cortical neurons were inhibited by repetitive VNS (pulses 50–150 μA, 0.5 ms, frequency 10 Hz). 7-NI administration did not change the amygdala-evoked neuronal reactions but reversed the effect of VNS on them. The present results suggest that the inhibitory action of VNS on amygdala–mPFC neurotransmission may involve a cortical NO-dependent mechanism.

The facilitating effect of systemic administration of Kv7/M channel blocker XE991 on LTP induction in the hippocampal CA1 area independent of muscarinic activation

A large amount of in vitro studies demonstrate suppression of M-current in hippocampal neurons by Kv7/M channel blocker results in depolarization of membrane potential and release of neurotransmitters, such as acetylcholine and glutamate, suggesting that Kv7/M channel may play important roles in regulating synaptic plasticity. In the present study, we examined the in vivo effect of Kv7/M channel inhibition on the long-term potentiation (LTP) induction at basal dendrites in hippocampal CA1 area of urethane-anaesthetized rats. The Kv7/M channel was inhibited by intraperitoneal injection of XE991 (10 mg/kg) and the LTP of field excitatory postsynaptic potential (fEPSP) was induced by supra-threshold high frequency stimulation (S1 HFS). A weak protocol which was just below the threshold for evoking LTP was used as sub-threshold high frequency stimulation (S2 HFS). XE991 did not significantly alter the slope of fEPSP and the magnitude of LTP induced by S1 HFS, suggesting that Kv7/M channel inhibition had little or no effect on glutamatergic transmission under basal conditions. However, XE991 could make S2 HFS evoke LTP even after the application of the muscarinic cholinergic (mACh) receptor antagonist scopolamine, suggesting that Kv7/M channel inhibition lowered the threshold for LTP induction and the effect was independent of muscarinic activation. Based on the above findings, we concluded that the facilitating effect of XE991 on LTP induction is not mediated by its ability to enhance the release of acetylcholine; therefore, Kv7/M channel blockers may provide a therapeutic benefit to cholinergic deficiency-related cognitive impairment, e.g., Alzheimer's disease.

Difference between male and female rats in cholinergic activity of parasympathetic vasodilatation in the masseter muscle

We compared the changes in blood flow of the masseter muscle (MBF), lower lip (LBF) and common carotid artery (CCABF) evoked by electrical stimulation of the lingual nerve (LN) in order to examine whether high cholinergic activity of parasympathetic vasodilatation in females is specific for the masseter muscle, and whether sex-associated differences in cholinergic parasympathetic vasodilatation affect the regulation of blood flow to the orofacial area from the CCABF in urethane-anaesthetized, vago-sympathectomized male and female rats. Increases in the MBF, LBF and CCABF evoked by LN stimulation appear to be mediated via an activation of parasympathetic reflex vasodilatation since these increases were profoundly reduced by pretreatment with the autonomic cholinergic ganglion blocker hexamethonium (10 mg/kg). Although α- and β-adrenoceptor antagonists (phentolamine and propranolol, 100 μg/kg) had no effect on the LN stimulation-induced blood flow increases in either sex, a marked difference was found between males and females in the effects of the antimuscarinic agent atropine (1–100 μg/kg) on these blood flow increases. Pretreatment with atropine slightly attenuated the increase in the MBF in males, but in females it markedly reduced the increases in all three sites measured, especially in the MBF. Our results suggest that (1) cholinergic activity of the parasympathetic vasodilatation in females is higher than that in males in most orofacial tissues, but particularly in the masseter muscle and (2) cholinergic parasympathetic vasodilatations are more involved in the regulation of blood flow to the orofacial area from the CCABF in females than in males.

Antagonism of glutamate receptors in the CA1 to perirhinal cortex projection prevents long-term potentiation and attenuates levels of brain-derived neurotrophic factor

The CA1 to perirhinal cortex projection is one of multiple hippocampal–neocortical projections considered to be involved in memory consolidation. This projection has been shown to sustain long-term potentiation (LTP) following stimulation of CA1. Here we examined the pharmacological properties underpinning the plasticity observed in this projection. A stimulating electrode was inserted into the area CA1 and a recording electrode was inserted into the perirhinal cortex of urethane-anaesthetised Wistar rats. Rats ( n = 6 in each drug group) were administered with either saline (0.09%), MK-801 (NMDA antagonist; 0.1 mg/kg) or CNQX (AMPA/kainate antagonist; 1.5 mg/kg). Baseline recordings were made for 10 min by stimulating area CA1 (0.05 Hz stimulation protocol). High-frequency stimulation (HFS; 250 Hz) was performed and post-HFS fEPSP recordings were made for 1 h (0.05 Hz, as above). Baseline and post-HFS paired-pulse facilitation (PPF) recordings were performed across six different interpulse intervals. CA1 and perirhinal cortex tissue samples were taken from the stimulated and unstimulated hemispheres of each rat brain and analysed using a brain-derived neurotrophic factor (BDNF) ELISA. Results indicate that LTP was induced in the saline and MK-801 groups but not in the CNQX group; fEPSPs in the latter group rapidly returned to baseline levels following a short period of post-tetanic potentiation. Drug treatment and HFS had no effect on PPF levels. Drug treatment significantly reduced concentrations of both CA1 and perirhinal BDNF and prevented stimulation-induced increases in BDNF in CA1. This molecular and electrophysiological data suggests that LTP in the CA1–perirhinal cortex projection may require activation of postsynaptic AMPA/kainate receptors in order to sustain LTP.

Characterization in vivo of bilaterally branching pontocerebellar mossy fibre to Golgi cell inputs in the rat cerebellum

Golgi cells regulate the flow of information from mossy fibres to the cerebellar cortex, through a mix of feedback and feedforward inhibitory actions on granule cells. The aim of the current study was to examine mossy fibre input to Golgi cells, in order to assess their impact on switching Golgi cells into feedforward behaviour. In urethane-anaesthetized rats, extracellular recordings were made from Golgi cells in Crus II (n = 18). Spikes were evoked in all Golgi cells by microstimulation within the contralateral hemispheral cortex, via branches of mossy fibres that terminate in both cerebellar hemispheres. The latencies of these responses were very short, consistent with a monosynaptic mossy fibre contact [average onset latency 2.3 +/- 0.1 ms (SEM)]. The same stimuli had no measurable effect on spike responses of nearby Purkinje cells (n = 12). Systematic mapping in the contralateral cerebellar hemisphere (Crus Ib, IIa, IIb and the paramedian lobule) usually revealed one low-intensity stimulus 'hotspot' (12-35 microA) from which short-latency spikes could be evoked in an individual Golgi cell. Microinjections of red and green retrograde tracers (latex beads, approximately 50-150 nL injection volume) made at the recording site and the stimulation hotspot resulted in double-labelled neurons within the pontine nuclei. Overall, this suggests that subsets of pontine neurons supply mossy fibres that branch to both hemispheres, some of which directly target Golgi cells. Such an arrangement may provide a common feedforward inhibitory link to temporally couple activity on both sides of the cerebellum during behaviour.

Inhibitory effects of ascaris suum extract On gastric acid secretion in rats

The effects of Ascars Suum (A. Suum) extract on gastric acid secretion were investigated in urethane-anaesthetised rats. Three determinations were done viz (1) the acid content when no drug was administered (2) the acid content when histamine was injected and (3) the acid content when the A. Suum extract was given alone or after histamine was administered. The extract (5.5 or 14mg/kg) reduced histamine-induced gastric acid secretion to the control value. At 14mg/kg the extract also reduced basal gastric acid secretion. At the peak of histamine-induced gastric acid secretion, the administration of the extract promptly reduced the gastric acid secretion to basal values. These results indicate that extracts of Ascaris Suum inhibit gastric acid secretion.

Copeptin Immunoreactivity and Calcium Mobilisation in Hypothalamic Neurones of the Rat

Copeptin is cleaved from the C-terminus of vasopressin (VP) prohormone. Immunohistochemical studies have revealed intense copeptin-immunoreactivity (irCOPT) in neurones of the rat hypothalamic nuclei, including paraventricular, supraoptic, suprachiasmatic, periventricular, and accessory secretory. Varicose cell processes emanated from irCOPT neurones, some of which projected caudally and traversed the internal layer of the median eminence, and terminated in the posterior pituitary. Double-labelling hypothalamic sections with copeptin antiserum and VP or oxytocin antiserum revealed an extensive overlapping of irCOPT and irVP neurones. The biological activity of human synthetic nonglycosylated copeptin or VP was evaluated in vivo and in vitro. Copeptin (1, 10, and 20 nmol/kg) injected i.v. caused no significant changes in the mean arterial pressure (MAP) and heart rate of urethane-anaesthetised rats. VP (0.1 nmol/kg) increased MAP, which was accompanied by a small decrease of the heart rate. The ratiometric fluorescence method was employed to assess changes in intracellular Ca2+ concentrations [Ca2+](i) which served as an index of the biological activity of peptides. VP (1 microM) markedly increased [Ca2+](i) of rat hypothalamic neurones or vascular smooth muscle cells, whereas copeptin (100 nm to 1 microM) caused a low amplitude, sustained increase of [Ca2+](i) in a population of hypothalamic neurones, but not in any of the vascular smooth muscle cells tested. The results obtained demonstrate that copeptin is expressed in VP neurones and that the peptide in the concentrations tested, although causing little or no detectable changes of blood pressure and heart rate in anaesthetised rats nor changes in [Ca2+](i) of cultured aortic smooth muscle cells, increases [Ca2+](i) in a small population (< 2%) of hypothalamic neurones tested, indicating that copeptin is biologically active in mammalian neurones.

Potentiation of reflex erectile responses in the anaesthetized rat by the selective melanocortin receptor 4 agonist MB243

To assess the potentiation of erectile responses induced by electrical stimulation (ES) of the dorsal penile nerve (DPN) in the urethane-anaesthetized rat by the selective melanocortin receptor 4 agonist MB243. Intracavernosal and blood pressures (ICP and BP, respectively) were monitored in urethane-anaesthetized rats after complete spinal cord transection at the thoracic level. Erectile responses were induced by ES of the DPN (train of square wave pulses of 1 ms and 6 V for 20 s at 1, 2 and 5 Hz) after i.v. injection of either saline or MB243, 3 mg/kg, in two groups of six rats. The maximal and mean ICP, and the area under the curve (AUC) of the ICP response, corrected for the corresponding BP, were measured and used as an index of erectile function (ICPmax/BP, ICPmean/BP and AUC/BP, respectively). MB243 increased the number of spontaneous erections between the injection and the first ES when compared with the vehicle group, but this difference was not statistically significant. ES of the DPN induced frequency-dependent erectile responses, the mean (sem) ICPmean/BP was 0.26 (0.02), 0.34 (0.04) and 0.39 (0.05) after administration of saline (vehicle) at 1, 2 and 5 Hz, respectively. All the variables, except the ICPmax/BP at 5 Hz, were significantly increased in the group injected with MB243 when compared with the vehicle group (P < 0.05 for ICPmax/BP and ICPmean/BP; P < 0.01 for AUC/BP). The AUC/BP showed the greatest increases of (+79%, +60% and +44% at 1, 2 and 5 Hz, respectively) in the group injected with MB243 compared with the vehicle group. Erectile responses induced by ES of the DPN in spinalized, urethane-anaesthetized rat are suitable for evaluating the proerectile facilitator activity of selective peripherally restricted melanocortin receptor 4 agonists. This model represents a valuable alternative to the classically used cavernous/pelvic nerve stimulated model.