Spinal vs. supraspinal sites of action of the alpha 2-adrenergic agonists clonidine and ST-91 on the acoustic startle reflex

Previous work has shown that stimulation of alpha 2-adrenergic receptors depresses the startle responses in rats. The present study suggests that this depressant effect involves supraspinal rather than spinal alpha 2-adrenergic receptors because intraventricular but not intrathecal infusion of the hydrophilic alpha 2-adrenergic agonist ST-91 depressed the acoustic startle reflex. To determine the point in the acoustic startle pathway where alpha 2-adrenergic receptor activation might ultimately alter neural transmission, startle responses were elicited electrically from different points along the acoustic startle pathway after systemic administration of clonidine. Clonidine depressed acoustically-elicited startle and startle elicited by electrical stimulation of the ventral cochlear nucleus to a comparable magnitude and over a similar time course. It also partially depressed startle elicited by electrical stimulation of the nucleus reticularis pontis caudalis (RPC). Taken together, these data suggest that alpha 2-adrenergic stimulation depresses startle by acting on supraspinal receptors, but that this effect is ultimately expressed, at least in part, by actions at both spinal and brainstem levels of the acoustic startle response pathway. The results are compared to other drugs known to affect the startle reflex.     

Preliminary evidence for an involvement of the cholinergic system in the sedative effects of rolipram in rats.

Rolipram is a specific cAMP phosphodiesterase type 4 (PDE4) inhibitor in the brain, which induces an increase in the intracellular levels of cAMP. Rolipram produces characteristic alterations in animal behavior, which have been suggested to be mediated mainly through an intracellular mechanism involving an increase in cAMP. However, specific mechanisms mediating the sedative effects of this compound have not yet been investigated. Because several lines of evidence indicate that the acetylcholine neural system may be involved in some effects of PDE4 inhibitors, the aim of this study was to elucidate whether the neurotransmitter acetylcholine is involved in the sedative effects induced by rolipram. The present study assessed the motor effects of rolipram in an exploratory behavioral test, the open field, in Wistar rats. The results show that rolipram (0.1-3.0 mg/kg SC) induced potent and dose-dependent hypoactivity, decreasing both locomotion and rearing. Physostigmine (0.03-0.3 mg/kg SC) potentiated a subeffective dose of rolipram (0.03 mg/kg SC), resulting in strong sedation, similar to that following higher doses of either rolipram or physostigmine alone, whereas the reduction in locomotor activity induced by rolipram (0.3 mg/kg SC) was completely reversed by scopolamine (0.03-0.3 mg/kg SC). These data provide preliminary evidence suggesting the involvement of the acetylcholinergic system in the sedative effects of rolipram.     

Developmental changes in pharmacological responsivity of the acoustic startle reflex: Effects of picrotoxin

Using the acoustic startle reflex as the behavioral measure, qualitatively different responses to the GABA antagonist picrotoxin were obtained in developing rats before and after 21 days postnatal (PN) age. Dose-dependent increases in acoustic startle were seen following picrotoxin in PN day 15-16 rat pups. In contrast, dose-dependent decreases in startle following picrotoxin were observed in adult rats. The switch from excitation to inhibition of startle was found to occur abruptly on PN day 21. Excitatory responses to picrotoxin were also found in adult rats following localized infusions of picrotoxin into lumbar spinal cord regions, but not into the forebrain. These results give evidence that picrotoxin-sensitive sites that modulate increases in startle reflex behavior mature first and are analogous to sites in the adult spinal cord, whereas picrotoxin-sensitive sites that modulate decreases in startle reflex behavior mature later (greater than or equal to PN day 21) and are localized in more rostral brain areas.     

Existence of rolipram-sensitive phosphodiesterase in rat megakaryocyte.

1. The effect of rolipram (ME3176) on ADP- and IP3-induced repetitive IK(Ca) in rat megakaryocyte was investigated by use of the nystatin perforated patch and conventional whole-cell patch-clamp techniques. 2. The ADP-induced IK(Ca) was depressed by treatment with rolipram in a concentration-dependent manner. The inhibition by rolipram disappeared after treatment with a cyclic nucleotide-dependent protein kinase inhibitor, H-8. The inhibition of IK(Ca) was also observed in the presence of cyclic AMP accumulating agents such as forskolin and isobutylmethylxanthine (IBMX). 3. Rolipram enhanced the inhibitory action of forskolin, suggesting that rolipram facilitates the accumulation of cyclic AMP by blocking its breakdown. Similar results was obtained with adenosine, an endogenous adenylate cyclase activator. 4. Intracellular application of inositol trisphosphate (IP3) induced repetitive IK(Ca) in megakaryocytes. The induced IK(Ca) was also inhibited by rolipram and by other cyclic AMP accumulating agents. 5. It was concluded that megakaryocytes possess rolipram-sensitive phosphodiesterase (PDE), which was not detected in platelets, but plays a distinct modulatory role in megakaryocytes for generating ADP-induced IK(Ca).     

Effects of rolipram on scopolamine-induced impairment of working and reference memory in the radial-arm maze tests in rats

RATIONALE: Rolipram, a selective inhibitor of cyclic AMP-specific phosphodiesterase (PDE4), has been shown to enhance scopolamine-induced impairment of working memory. However, its effect on reference memory, which appears to be related to the level of cyclic AMP (cAMP), has not been investigated yet; in addition, the mechanism involved in its effects on memory remains to be elucidated. OBJECTIVES: To investigate the effects of rolipram on working and reference memories impaired by scopolamine and the involvement of cAMP. METHODS: By administration (IP) of rolipram and forskolin, an activator of adenylyl cyclase (AC), the effects of both drugs on the number of correct choices and errors in experiment 1 and, the frequency of both working memory errors and reference memory errors in experiment 2 were observed in two eight-arm radial maze tasks in rats. RESULTS: In experiment 1, rolipram (0.01-1.0 mg/kg) attenuated the scopolamine-induced (0.5 mg/kg) increase in the total number of errors in dose- and time-dependent manners. The minimum effective dose of rolipram was 0.05 mg/kg and the effects lasted nearly 60 min. By contrast, forskolin (1.0-10.0 mg/kg) failed significantly to affect any of the above indices altered by scopolamine. In experiment 2, rolipram (0.05 and 0.1 mg/kg) decreased the frequencies of both working and reference memory errors that were elevated by scopolamine. Forskolin did not alter either type of error at a dose that increased the exploration time. CONCLUSION: Rolipram may exert its effects of reversing both working and reference memory impairments via increased cyclic AMP concentrations in certain signal transduction pathways, rather than by a generalized increase in cAMP.     

Potential antidepressant activity of rolipram and other selective cyclic adenosine 3',5'-monophosphate phosphodiesterase inhibitors

Following intraperitoneal administration, the selective cAMP phosphodiesterase (PDE) inhibitors rolipram, ICI 63 197 and Ro 20-1724 were investigated in mice in comparison with imipramine for their effectiveness in two classical test models for prediction of clinical antidepressant activity: antagonism of reserpine-induced hypothermia or hypokinesia and potentiation of yohimbine lethality. Rolipram was approximately 15 times more potent than imipramine or Ro 20-1724 and approximately as potent as ICI 63 197 in antagonizing reserpine-induced hypothermia. The antihypothermic effect of the phosphodiesterase inhibitors occurred at a smaller dose than that of imipramine. In contrast to imipramine, the phosphodiesterase inhibitors reversed reserpine-induced hypokinesia. Rolipram was approximately as potent as ICI 63 197 and about 15 times more potent than Ro 20-1724. Rolipram was approx. 5 times more potent than Ro 20-1724 and approx. as potent as imipramine or ICI 63 197 in potentiating the lethality of yohimbine. In both test models the (-)-isomer of rolipram was approx. 10-15 times more potent than the (+)-isomer, indicating a stereospecific mechanism of action. The present data suggest an antidepressant action of selective cAMP phosphodiesterase inhibitors due to enhancement of central noradrenergic transmission. The hypothesis is put forward that the increase of noradrenaline turnover induced by phosphodiesterase inhibitors in conjunction with the inhibitory action of the compounds on cAMP metabolism enables more efficient adaptative changes to occur at central synapses resulting in a rapid onset of the antidepressant activity. Preliminary results with rolipram in patients with endogenous depression seem to support this assumption.     

Diethyldithiocarbamate depresses the acoustic startle response in rats

The relationship between norepinephrine (NE) content in cortex and spinal cord and acoustic startle amplitude was investigated in two experiments. Administration of diethyldithiocarbamate (DDC) depressed startle amplitude at the same time and dose that it most severely depleted NE content. These results support the conclusion that NE facilitates the normal elaboration of the acoustic startle reflex and also support evidence that NE activity in the spinal cord may be of particular importance in the maintenance of normal startle amplitude.     

Effects of the cyclic nucleotide phosphodiesterase inhibitors, rolipram, 3-isobutyl-1-methylxanthine, amrinone and zaprinast, on pancreatic exocrine secretion in dogs.

The effects of the cyclic phosphodiesterase (PDE) inhibitors, rolipram, 3-isobutyl-1-methylxanthine (IBMX), amrinone and zaprinast on pancreatic exocrine secretion were investigated in anesthetized dogs. Rolipram (1-30 nmol), IBMX (44-440 nmol) or zaprinast (1-10 mumol) injected i.a. elicited a dose-dependent increase in the secretion of pancreatic juice, but amrinone (up to 53 mumol) did not. The bicarbonate concentration in pancreatic juice was increased and the protein concentration was decreased by rolipram and IBMX, but neither was affected by zaprinast. Rolipram elicited more than the respective additive secretory response when added together with secretin, although the stimulatory effects of CCK-8 with rolipram were additive. Rolipram and IBMX, but not zaprinast, increased cyclic AMP concentration but did not affect cyclic GMP concentration. These results suggest that rolipram, IBMX and zaprinast have direct secretory properties on pancreatic exocrine glands of the dog, which may be mediated through the increase of intracellular cyclic AMP concentration, by inhibiting PDE activity. Furthermore, the pancreatic PDE enzyme in the dog pancreas may be mainly a type IV.     

Stereospecific binding of the antidepressant rolipram to brain protein structures

The characteristics for the binding of the selective cAMP phosphodiesterase inhibitor and antidepressant agent rolipram to brain and peripheral organs were investigated. (+/-)-[3H]Rolipram equilibrium binding and Scatchard analysis revealed saturable, reversible, stereospecific, Mg2+-dependent and heat-sensitive binding with an apparent Hill number of 1. Binding was detected both to membrane-bound and soluble sites, with dissociation constants Kd of 1.2 and 2.4 nM, respectively, and binding site concentrations (Bmax) of 19.3 and 23.6 pmol/g rat forebrain. The (-)-enantiomer of rolipram was ca. 20 times more effective than the (+)-enantiomer in displacing (+/-)-[3H]rolipram from membranes. Rolipram bound to brain tissue of all mammalian species tested including man, while tissue from bird and fish showed less binding. Organs other than brain exhibited only negligible binding. Only specific cAMP phosphodiesterase inhibitors (ICI 63.197, Ro 20-1724) were potent competitors, while rolipram itself was inactive in a variety of receptor binding assays of neuroactive ligands. The kinetics of (-)-[3H]rolipram binding to the particulate fraction revealed a complex association and dissociation behaviour. The nature of the rolipram binding protein(s) is not clear, but the low affinity binding site evident from binding kinetics may represent a rolipram-sensitive phosphodiesterase isoenzyme also common to some peripheral organs, while the high affinity binding site(s) may be related to PDE isoenzymes more confined to the central nervous system.     

Anti-inflammatory and analgesic effects of the phosphodiesterase 4 inhibitor rolipram in a rat model of arthritis

There has been much interest in strategies which modulate tumour necrosis factor-alpha (TNF-alpha) levels and/or function in rheumatoid arthritis. The elevation of intracellular levels of cyclic AMP in leukocytes by phosphodiesterase 4 inhibitors is accompanied by significant inhibition of the production of TNF-alpha. Nevertheless, these drugs may enhance the hyperalgesia induced by a range of inflammatory mediators, including TNF-alpha. In the present study, we examined the effects of the phosphodiesterase 4 inhibitor rolipram on the local inflammatory infiltrate and hyperalgesia in a rat model of adjuvant-induced arthritis. Rolipram (3 mg/kg) was administered by oral gavage from day 10 to 14 after disease induction. Pretreatment with rolipram abrogated oedema formation and significantly inhibited hyperalgesia. Histopathological analysis revealed a marked inhibition of cellular influx as well as bone and cartilage destruction. Serum and local TNF-alpha levels were suppressed in treated animals whereas there were little changes in interleukin-1beta levels. Although cyclic AMP elevating agents may affect nociceptor threshold to increase the hyperalgesic responses acutely, they also possess significant anti-inflammatory activity, which may hinder local mediator release and/or action. The anti-inflammatory effects of rolipram predominate during this chronic arthritis model in the rat.     

Electroencephalographic study with rolipram (ME 3167), a selective inhibitor of adenosine 3',5'-monophosphate phosphodiesterase, in rabbits]

The electroencephalographic (EEG) effects of rolipram were investigated in rabbits with chronic electrode implants. Intravenous administration of rolipram at 10 to 100 micrograms/kg evoked an increase in the arousal EEG pattern period (low amplitude fast waves) in the cortical EEG and synchronization of the hippocampal theta waves with increased voltages. Rolipram at higher doses caused behavioral excitation in rabbits. Rolipram did not affect EEG arousal responses to both auditory stimulation (2000 Hz, monotone) and electrical stimulation (100 Hz, 0.1 msec, 3-6 V) of the midbrain reticular formation or the posterior hypothalamus. The recruiting response induced by centromedian thalamic stimulation at a low frequency (7 Hz, 0.1 msec, 4-8 V) was not altered by rolipram. Rolipram was without effect on the photic driving response to flash light (2 Hz) in the occipital cortex. Rolipram caused a slight increase in the duration of after discharges induced by electrical stimulation (50 Hz, 0.5 msec, 4-20 V) of the dorsal hippocampus. These results suggest that rolipram is a drug that causes the EEG arousal pattern.     

Hippocampal modulation of acoustic startle and prepulse inhibition in the rat.

Prepulse inhibition (PPI) is the normal reduction in a startle response that occurs when the startling stimulus is preceded by a weak lead stimulus ("prepulse"). Schizophrenic patients exhibit abnormally low levels of PPI; therefore, animal models of deficient PPI may provide information regarding neural dysfunctions underlying schizophrenia. We recently reported that infusion of the cholinergic agonist carbachol into the dentate gyrus (DG) disrupts PPI in the rat. We now report the effects of carbachol microinjected into CA1, the DG, or the ventral subiculum (VS) on acoustic startle and PPI. Carbachol infusion into CA1 or the DG depressed startle. Carbachol infusion decreased PPI with a regional rank-order potency CA1 > DG > VS. CA1 infusions more potently depressed the startle reflex. By contrast, DG infusions preferentially decreased PPI, while VS infusions decreased PPI without altering startle amplitude. Coinfusion with the muscarinic cholinergic antagonist atropine opposed the effects of carbachol. These results demonstrate the regional heterogeneity and pharmacological specificity of the hippocampal cholinergic modulation of acoustic startle and PPI and suggest that abnormalities within various regions of the hippocampal formation may contribute to deficient sensorimotor gating in schizophrenic patients.     

Effects of lithium and rolipram enantiomers on locomotor activity in inbred mice

Rolipram is a racemic drug that increases brain cAMP availability by inhibiting phosphodiesterase. Lithium, on the other hand, reduces cAMP levels in brain by inhibiting adenylate cyclase and by increasing phosphodiesterase activity. Inbred strains of mice were used in the present study to determine whether the opposite effects of lithium and rolipram on cAMP levels were evident from behavioural effects of the drugs. In the first experiment, locomotor activity was measured in strains of male and female mice given an injection of either vehicle, (+)- or (-)-rolipram in order to find appropriate test conditions for studying interactions with lithium. (-)-Rolipram was more potent than its antipode in inducing hypokinesia, and the effects of (+)- and (-)-rolipram depended on mouse strain. In the second experiment, mice received lithium in their food for 3-4 weeks prior to testing, and they were then given an injection of vehicle, (+)- or (-)-rolipram before behaviour tests. Lithium reduced locomotor activity in mice given the vehicle, whereas it counteracted activity-suppressant effects of rolipram. The behavioural findings are consistent with the opposite pharmacological effects of lithium and rolipram on cAMP, and support the notion that cAMP availability in brain may play a role in behavioural effects of lithium and rolipram.     

Effects of pyrethroids on the acoustic startle reflex in the rat

The effects of NAK 1901 (Pentafluorbenzyl (1R, cis)-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropane-carboxylate) and cypermethrin ((S,R)-alpha-cyano-3-phenoxybenzyl-2,2-dimethyl (1R, 1S, cis, trans)-3-(2,2-dichlorovinyl) cyclopropane-carboxylate) (RU 24 501) on amplitude and prepulse inhibition of the acoustic startle reflex were studied in male Wistar rats. NAK 1901 (0, 1, 2.5, 4 mg/kg p.o.) enhanced the amplitude of the startle reflex in a dose-dependent way. Startle latency was not affected. Cypermethrin (0, 0.5, 1, 2 mg/kg p.o.) had no effect on the amplitude or the latency of the startle reflex. Both NAK 1901 and cypermethrin administration produced a dose-dependent increase in toxic signs and a dose-dependent increase in weight loss during the experimental session. None of the pyrethroids affected prepulse inhibition of the startle reflex. Because the neural substrate of the inhibitory processes involved in prepulse inhibition are probably of supraspinal origin, it is suggested that these substrates are not affected by pyrethroids.     

Effect of clonidine on the human acoustic startle reflex

The present study investigated in healthy human volunteers whether clonidine reduced the amplitude of the acoustic startle reflex and whether this effect, if found, was due to an accelerated rate of habituation. Subjects were presented with startleeliciting noise-bursts after intravenous (iv) infusion of clonidine (1.5 µg/kg) and saline on separate days. Clonidine significantly reduced the amplitude of the acoustic startle reflex (as indexed by the eyeblink component) relative to the saline treated condition. This effect was neither due to an accelerated rate of habituation of the startle reflex nor to the sedative effect of clonidine. These findings complement an earlier report (Morgan et al. 1993) that yohimbine augments the amplitude of the startle reflex in man. Taken together, the two reports indicate a new model for the clinical investigation of central alpha₂ adrenoceptor function in humans.     

Central serotonin involvement in the elaboration of the startle reaction in rats

The present investigation provides evidence for serotonergic involvement in the inhibition of the acoustic startle reaction which results from the presentation of neutral stimuli (prepulses) shortly before reflex elicitation. While the specific serotonin (5-HT) synthesis inhibitor p-Chlorophenylalanine did not affect the response, a large amine-depleting dose of reserpine enhanced the reaction elicited under stable (control) stimulus conditions (no prepulse delivered). Selective replacement of 5-HT by administration of the l-amino acid precursor and a monoamine oxidase inhibitor further enhanced control startle amplitude and also eliminated prepulse inhibition. Chemical assays indicated especially large increases in 5-HT levels in the cortex and brainstem. Administration of the 5-HT precursor in the non-reserpinized rat both increased control startle levels and also interfered with prepulse inhibition although not as completely as in the reserpinized animal. The results indicate that 5-HT has a facilitatory effect on the reflex and suggest that a catecholaminergic system is also involved in determining reflex amplitude.     

Effect of methamphetamine on rat spinal cord. Dopamine receptor-mediated depression of monosynaptic reflex

The experiments were performed on spinal rats transected at Cl. Intravenous administration of methamphetamine-HCl (MA-HCl, 2 mg/kg) and apomorphine-HCl (5 mg/kg) reduced the amplitude of the monosynaptic reflex (MSR), while the polysynaptic reflex was increased by methamphetamine. Depression of the monosynaptic reflex by both drugs was antagonized by haloperidol, but not by phentolamine. Depression of the monosynaptic reflex by methamphetamine was not antagonized by pretreatment with reserpine; however, the result was explained by the assumption that methamphetamine releases newly-synthesized dopamine or that methamphetamine may act directly on dopamine receptors. Depression of the monosynaptic reflex induced by methamphetamine was independent of peripheral changes in blood pressure. Oxygen tension in the spinal cord was slightly reduced by methamphetamine in rats treated with phentolamine and a change of pO2 in the spinal cord was ruled out as a possible mechanism of action. These results suggest that dopaminergic neurons in the spinal cord of the rat depress the transmission of monosynaptic spinal reflexes.     

Antidepressant properties of some phosphodiesterase inhibitors

Effects of three phosphodiesterase (PDE) inhibitors (rolipram, IBMX, and Ro 20-1724) were studied in mice in some tests predictive for antidepressant activity. All the drugs antagonized reserpine-induced hypothermia and slightly antagonized reserpine-evoked hypoactivity. Rolipram and IBMX were also effective in behavioral despair test. On the other hand, the examined PDE inhibitors did not affect apomorphine-induced hypothermia and did not potentiate the central action of L-DOPA. These results show that psychopharmacological profile of the PDE inhibitors differs from that of classical imipramine-like antidepressants.     

The modification of lumbar motoneurone excitability by stimulation of a putative 5-hydroxytryptamine pathway

1 Changes in lumbar motoneurone excitability were monitored by recording spinal reflex activity from the ventral roots of rats anaesthetized with fluothane.2 Electrical stimulation of nucleus raphes medianus increased the amplitude of the monosynaptic reflex via a pathway having a slow conduction velocity. Stimulation elsewhere in the lower brain stem was less effective. This increase in motoneurone excitability was potentiated by the intravenous injection of L-tryptophan and reduced by intravenous injections of lysergic acid diethylamide (LSD), methysergide or Cinanserin.3 Extracellular field potential responses to stimulation of dorsal or ventral roots were recorded with six barrelled microiontophoresis electrodes. Stimulation of nucleus raphes medianus and iontophoretic application of 5-hydroxytryptamine (5-HT) both increased the excitability of lumbar motoneurones as reflected by an increase in field potential amplitude.4 Responses to both stimulation of raphe nuclei and iontophoretic application of 5-HT were reduced by iontophoretic application of Cinanserin and methysergide.5 The similarities of the responses of lumbar motoneurones to applied 5-HT and activity within the raphe-spinal pathway are discussed. It is suggested that activity within the raphe-spinal pathway can increase lumbar motoneurone excitability via the release of 5-HT in the ventral horn of the spinal cord.     

Lesions of the laterodorsal tegmental nucleus disrupt prepulse inhibition of the acoustic startle reflex

The purpose of the present studies was to determine the effects of bilateral lesions of the laterodorsal tegmental nucleus (LDTg) on prepulse inhibition (PPI) of the acoustic startle reflex under conditions of varying prepulse intensity and interstimulus interval (ISI) durations. Rats with bilateral ibotenic acid lesions of the LDTg were evaluated for changes in PPI and startle amplitude in comparison with an unoperated group, sham-LDTg lesioned group and a group with bilateral ibotenic acid lesions of the subcoeruleus nucleus, a brainstem nucleus approximately 1 mm ventrolateral to the LDTg. Bilateral lesions of the LDTg produced a robust decrease in PPI with no effect on startle amplitude as compared with the three control groups. In contrast, bilateral lesions of the subcoeruleus produced no effect on either PPI or startle amplitude. The effects of bilateral lesions of the LDTg on PPI were observed across prepulse intensities of 5, 10 and 15 dB above background and ISI durations of 30, 100, 300 and 1000 ms without significantly decreasing startle amplitude in either test paradigm as compared with the sham-LDTg lesioned group. Our data provide evidence for a role of the LDTg in modulating PPI.