DSP-4 Pretreatment Research Articles

Opposing effects of β-2 and β-1 adrenergic receptor signaling on neuroinflammation and dopaminergic neuron survival in α-synuclein-mediated neurotoxicity

BackgroundNoradrenergic neurons in the locus coeruleus (LC) are the primary source of norepinephrine (NE) in the brain and degeneration of these neurons is reported in the early stages of Parkinson’s disease (PD), even prior to dopaminergic neuron degeneration in the substantia nigra (SN), which is a hallmark of PD pathology. NE depletion is generally associated with increased PD pathology in neurotoxin-based PD models. The effect of NE depletion in other models of PD-like α-synuclein-based models is largely unexplored. In PD models and in human patients, β-adrenergic receptors’ (AR) signaling is associated with a reduction of neuroinflammation and PD pathology. However, the effect of NE depletion in the brain and the extent of NE and β-ARs signaling involvement in neuroinflammation, and dopaminergic neuron survival is poorly understood.MethodsTwo mouse models of PD, a 6OHDA neurotoxin-based model and a human α-synuclein (hα-SYN) virus-based model of PD, were used. DSP-4 was used to deplete NE levels in the brain and its effect was confirmed by HPLC with electrochemical detection. A pharmacological approach was used to mechanistically understand the impact of DSP-4 in the hα-SYN model of PD using a norepinephrine transporter (NET) and a β-AR blocker. Epifluorescence and confocal imaging were used to study changes in microglia activation and T-cell infiltration after β1-AR and β2-AR agonist treatment in the hα-SYN virus-based model of PD.ResultsConsistent with previous studies, we found that DSP-4 pretreatment increased dopaminergic neuron loss after 6OHDA injection. In contrast, DSP-4 pretreatment protected dopaminergic neurons after hα-SYN overexpression. DSP-4-mediated protection of dopaminergic neurons after hα-SYN overexpression was dependent on β-AR signaling since using a β-AR blocker prevented DSP-4-mediated dopaminergic neuron protection in this model of PD. Finally, we found that the β-2AR agonist, clenbuterol, reduced microglia activation, T-cell infiltration, and dopaminergic neuron degeneration, whereas xamoterol a β-1AR agonist showed increased neuroinflammation, blood brain barrier permeability (BBB), and dopaminergic neuron degeneration in the context of hα-SYN-mediated neurotoxicity.ConclusionsOur data demonstrate that the effects of DSP-4 on dopaminergic neuron degeneration are model specific, and suggest that in the context of α-SYN-driven neuropathology, β2-AR specific agonists may have therapeutic benefit in PD.

Dexmedetomidine Ameliorated Cognitive Dysfunction Induced by Intestinal Ischemia Reperfusion in Mice with Possible Relation to the Anti-inflammatory Effect Through the Locus Coeruleus Norepinephrine System

Cognitive impairment is a common central nervous system complication that occurs following surgery or organs damage outside the nervous system. Neuroinflammation plays a key role in the molecular mechanisms of cognitive impairment. Dexmedetomidine alleviates neuroinflammation and reduces cognitive dysfunction incidence; however, the mechanism by which dexmedetomidine alleviates cognitive dysfunction remains unclear. This study evaluated the effect of dexmedetomidine on attenuation of early cognitive impairment induced by intestinal ischemia–reperfusion in mice and examined whether the locus coeruleus norepinephrine (LCNE) system participates in the anti-inflammatory effect of dexmedetomidine. The superior mesenteric artery was clamped for 45 min to induce intestinal ischemia reperfusion injury. Dexmedetomidine alone or combined with DSP-4, a selective locus coeruleus noradrenergic neurotoxin, was used for pretreatment. Postoperative cognition was assessed using the Morris water maze. Serum and hippocampal levels of IL-1β, TNF-α, norepinephrine (NE), and malondialdehyde (MDA) were assessed by enzyme-linked immunosorbent assay. Immunofluorescence, immunohistochemistry, and hematoxylin and eosin staining were used to evaluate the expression of tyrosine hydroxylase (TH) in the locus coeruleus, hippocampal microglia, and intestinal injury. Pretreatment with dexmedetomidine alleviated intestinal injury and decreased the serum and hippocampal levels of NE, IL-1β, TNF-α, and MDA at 24 h after intestinal ischemia reperfusion, decreased TH-positive neurons in the locus coeruleus, and ameliorated cognitive impairment. Similarly, DSP-4 pre-treatment alleviated neuroinflammation and improved cognitive function. Furthermore, α2-adrenergic receptor antagonist atipamezole or yohimbine administration diminished the neuroprotective effects and improved cognitive function with dexmedetomidine. Therefore, dexmedetomidine attenuated early cognitive dysfunction induced by intestinal ischemia–reperfusion injury in mice, which may be related to its anti-inflammatory effects through the LCNE system.

Repeated Administration of Amitriptyline in Neuropathic Pain: Modulation of the Noradrenergic Descending Inhibitory System.

The tricyclic antidepressant amitriptyline, the serotonin and noradrenaline reuptake inhibitor duloxetine, and gabapentinoids are first-line drugs for treatment of neuropathic pain. The analgesic effect of these drugs relates to brainstem-spinal descending noradrenergic systems. However, amitriptyline utilizes a variety of mechanisms for analgesia in neuropathic pain, and it is unclear which mechanism is most important. In the present study, we investigated the role of descending noradrenergic systems in the analgesic effect of these drugs for treatment of neuropathic pain. We also examined whether amitriptyline modifies the descending noradrenergic systems. Seven days after L5 spinal nerve ligation (SNL), rats received N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, 50 mg/kg) to degenerate noradrenergic fibers. The rats then received 5 daily intraperitoneal injections of amitriptyline (10 mg/kg), duloxetine (10 mg/kg), pregabalin (10 mg/kg), or gabapentin (50 mg/kg) from 21 days after SNL surgery. Paw withdrawal thresholds were determined to assess the effect of the drugs on hyperalgesia after SNL. To determine whether 5 daily injections of amitriptyline activated noradrenergic neurons in the locus coeruleus (LC) and spinal cord with or without DSP-4 treatment, we performed immunohistochemistry using antibodies for c-Fos and dopamine beta-hydroxylase (DβH). Five daily injections of amitriptyline, duloxetine, pregabalin, and gabapentin exerted antihyperalgesic effects in SNL rats (P < .001; estimated treatment effect of amitriptyline [99% confidence interval]: 59.9 [35.1-84.7] g). The antihyperalgesic effects of duloxetine, pregabalin, and gabapentin were reversed by pretreatment with DSP-4 (P < .001, respectively). However, antihyperalgesia was still observed after treatment of amitriptyline in SNL rats with DSP-4 pretreatment (P < .001, 59.7 [30.0-89.3] g), and this analgesic effect was not reversed by the α2-adrenoceptor antagonist idazoxan (30 μg). Additionally, 5 daily injections of amitriptyline increased the ratio of c-Fos-immunoreactive (IR) cells in noradrenergic LC neurons in SNL rats with or without DSP-4 pretreatment (P < .001, respectively). Five daily injections of amitriptyline increased DβH-IR in the LC and the spinal dorsal horn of SNL rats (P < .001, respectively). With DSP-4 pretreatment, DβH-IR was dramatically decreased with or without 5 daily injections of amitriptyline (P < .001). Five daily injections of amitriptyline produced antihyperalgesic effects against neuropathic pain despite suppression of noradrenergic descending inhibitory systems. Amitriptyline activated LC neurons and increased noradrenergic fibers density in SNL rats. These results suggest that amitriptyline could still produce analgesia under pathological dysfunction of the descending noradrenergic system. Amitriptyline may enhance the analgesic effect of drugs for neuropathic pain that require normal descending noradrenergic inhibition to produce analgesia, such as serotonin and noradrenaline reuptake inhibitors and gabapentinoids.

Monoamine involvement in the antidepressant-like effect induced by P2 blockade

Depression is a common mental disorder that affects millions of individuals worldwide. Available monoaminergic antidepressants are far from ideal since they show delayed onset of action and are ineffective in approximately 40% of patients, thus indicating the need of new and more effective drugs. ATP signaling through P2 receptors seems to play an important role in neuropathological mechanisms involved in depression, since their pharmacological or genetic inactivation induce antidepressant-like effects in the forced swimming test (FST). However, the mechanisms involved in these effects are not completely understood. The present work investigated monoamine involvement in the antidepressant-like effect induced by non-specific P2 receptor antagonist (PPADS) administration. First, the effects of combining sub-effective doses of PPADS with sub-effective doses of fluoxetine (FLX, selective serotonin reuptake inhibitor) or reboxetine (RBX, selective noradrenaline reuptake inhibitor) were investigated in mice submitted to FST. Significant antidepressant-like effect was observed when subeffective doses of PPADS was combined with subeffective doses of either FLX or RBX, with no significant locomotor changes. Next, the effects of depleting serotonin and noradrenaline levels, by means of PCPA (p-Chlorophenylalanine) or DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride) pretreatment, respectively, was investigated. Both, PCPA and DSP-4 pretreatment partially attenuated PPADS-induced effects in FST, without inducing relevant locomotor changes. Our results suggest that the antidepressant-like effect of PPADS involves modulation of serotonin and noradrenaline levels in the brain.

Depletion of norepinephrine of the central nervous system Down-regulates the blood glucose level in d-glucose-fed and restraint stress models

DSP-4[N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride] is a neurotoxin that depletes norepinephrine. The catecholaminergic system has been implicated in the regulation of blood glucose level. In the present study, the effect of DSP-4 administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) on blood glucose level was examined in d-glucose-fed and restraint stress mice models. Mice were pretreated once i.c.v. or i.t. with DSP-4 (10–40μg) for 3days, and d-glucose (2g/kg) was fed orally. Blood glucose level was measured 0 (prior to glucose feeding or restraint stress), 30, 60, and 120min after d-glucose feeding or restraint stress. The i.c.v. or i.t. pretreatment with DSP-4 attenuated blood glucose level in the d-glucose-fed model. Plasma corticosterone level was downregulated in the d-glucose-fed model, whereas plasma insulin level increased in the d-glucose-fed group. The i.c.v. or i.t. pretreatment with DSP-4 reversed the downregulation of plasma corticosterone induced by feeding d-glucose. In addition, the d-glucose-induced increase in plasma insulin was attenuated by the DSP-4 pretreatment. Furthermore, i.c.v. or i.t. pretreatment with DSP-4 reduced restraint stress-induced increases in blood glucose levels. Restraint stress increased plasma corticosterone and insulin levels. The i.c.v. pretreatment with DSP-4 attenuated restraint stress-induced plasma corticosterone and insulin levels. Our results suggest that depleting norepinephrine at the supraspinal and spinal levels appears to be responsible for downregulating blood glucose levels in both d-glucose-fed and restraint stress models.

Noradrenergic inputs from locus coeruleus to posterior ventral tegmental area are essential to support ethanol reinforcement.

Although dysregulation of the dopaminergic mesolimbic system is generally considered central to addiction, the involvement of other circuits is increasingly being appreciated. An interaction between locus coeruleus (LC) noradrenergic neurons and the posterior ventral tegmental area (pVTA) dopaminergic system, in the processing of drug-triggered reward, has been suggested, but not demonstrated in behaving animals. Herein, we try to tease out the precise role of noradrenergic neurons in the LC-VTA circuit in mediating reward and reinforcement behavior associated with ethanol. In the standard two-lever (active/inactive) operant paradigm, the rats were trained to self-administer ethanol in pVTA and subjected to pharmacological intervention. Intra-pVTA administration of phenylephrine (alpha-1 adrenoceptor agonist) increased ethanol self-administration, while prazosin and disulfiram (agents that reduce noradrenergic tone) produced opposite effects. While degeneration [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride, DSP-4, intraperitoneal route] or silencing (lidocaine or muscimol, both via intra-LC route) of the LC noradrenergic neurons decreased, phenylephrine via the intra-LC route reinstated ethanol self-administration. Furthermore, lidocaine reduced ethanol self-administration, but the effect was fully attenuated by noradrenaline given directly in the pVTA. This suggests that the feedback signals from LC to pVTA are necessary to sustain the ethanol self-infusion activity. Ethanol self-administration significantly increased tyrosine hydroxylase immunoreactivity in pVTA and LC; the response was blocked by DSP-4 pre-treatment. While dopamine D1 , but not D2 , receptors were localized on noradrenergic LC neurons, pre-treatment with SCH-23390 (intra-LC) dampened the lever press activity. We suggest that two-way communications between VTA and LC regions is essential for ethanol-triggered reinforcement behavior.

Noradrenergic-Dopaminergic Interactions Due to DSP-4-MPTP Neurotoxin Treatments: Iron Connection.

The investigations of noradrenergic lesions and dopaminergic lesions have established particular profiles of functional deficits and accompanying alterations of biomarkers in brain regions and circuits. In the present account, the focus of these lesions is directed toward the effects upon dopaminergic neurotransmission and expression that are associated with the movement disorders and psychosis-like behavior. In this context, it was established that noradrenergic denervation, through administration of the selective noradrenaline (NA) neurotoxin, DSP-4, should be performed prior to the depletion of dopamine (DA) with the selective neurotoxin, MPTP. Employing this regime, it was shown that (i) following DSP-4 (50mg/kg) pretreatment of C57/Bl6 mice, both the functional and neurochemical (DA loss) effects of MPTP (2×20 and 2×40mg/kg) were markedly exacerbated, and (ii) following postnatal iron (Fe(2+), 7.5mg/kg, on postnatal days 19-12), pretreatment with DSP-4 followed by the lower 2×20mg/kg MPTP dose induced even greater losses of motor behavior and striatal DA. As yet, the combination of NA-DA depletions, and even more so Fe(2+)-NA-DA depletion, has been considered to present a movement disorder aspect although studies exploring cognitive domains are lacking. With intrusion of iron overload into this formula, the likelihood of neuropsychiatric disorder, as well, unfolds.

Effects of atomoxetine on attention and impulsivity in the five-choice serial reaction time task in rats with lesions of dorsal noradrenergic ascending bundle.

Atomoxetine, a noradrenaline reuptake inhibitor (NRI), which is a non-stimulating medicine that is used for the treatment of patients with attention deficit hyperactivity disorder (ADHD), has been found to be effective in reducing behavioral impulsivity in rodents, but its efficacy in a dorsal noradrenergic ascending bundle (DNAB)-lesioned condition has not been examined. The present study aimed to investigate the effects of DNAB lesions on attention and impulsive control in the five-choice serial reaction time task (5-CSRTT) in rats treated with atomoxetine. The drug-induced changes in noradrenaline efflux in the medial prefrontal cortex were also measured. 5-CSRTT-trained rats were included in one of the following groups: N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)/Atomoxetine, Sham/Atomoxetine, DSP-4/Saline, or Sham/Saline. Acute atomoxetine (0.3 mg/kg) was administered 14 days after the DSP-4 regime. The behavioral testing included manipulations of the inter-trial interval (ITI), stimulation duration and food satiety. In vivo microdialysis of the noradrenaline efflux in the medial prefrontal cortex and the expression of the noradrenaline transporter (NAT) in the DNAB areas were examined. Atomoxetine reduced impulsivity and perseveration in the long-ITI condition with no effects on any other variables. This phenomenon was not influenced by DSP-4 pre-treatment. The DNAB-lesioned rats had lower noradrenaline efflux in the medial prefrontal cortex. DSP-4 caused no change in NAT expression in the DNAB areas. These findings suggested that noradrenaline reuptake may not be exclusively responsible for the atomoxetine effects in adjusting impulsivity. The role of DNAB should also be considered, particularly in conditions requiring greater behavioral inhibition.

The effect of denervation of the locus coeruleus projections with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) on cocaine-induced locomotion and place preference in rats

The potential contribution of locus coeruleus (LC)-derived noradrenaline (NA) in the motor activating and rewarding effects of cocaine (15 mg/kg) were assessed following administration of the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). In Experiment 1, administration of 10 mg/kg of DSP-4 similarly to substantial denervation with 50 mg/kg of DSP-4 significantly attenuated the activating effects of cocaine during the first cocaine-paired training session (30 min) in the conditioned place preference (CPP) apparatus. Only administration of the higher dose (50 mg/kg) of DSP-4 attenuated line crossings during the last training, while both doses reduced rearings. Thus, both minor and substantial denervation of LC reduced but did not abolish locomotion activating effect of cocaine. Cocaine CPP as measured by increment of time spent in the previously cocaine-paired chamber during drug-free conditions before and after cocaine-paired trainings was clearly revealed only in animals with intact projections from the LC, and was entirely absent after a large lesion of LC projections by DSP-4 (50 mg/kg). Because recovery of noradrenaline levels by the end of experiment did not allow assessment of the efficacy of the neurotoxin, the effect of DSP-4 pre-treatment on the acute psychomotor effect of cocaine was re-examined in an independent experiment (Experiment 2). Near complete denervation of the LC projections again reduced the effect of cocaine, but the lower dose of DSP-4 had no effect, suggesting that small lesions of the LC do not have a robust impact. Overall, this study demonstrates that both unconditioned and conditioned effects of cocaine depend upon the integrity of LC projections.

Central noradrenergic depletion by DSP-4 prevents stress-induced memory impairments in the object recognition task

Environmental stress produces adverse affects on memory in humans and rodents. Increased noradrenergic neurotransmission is a major component of the response to stress and noradrenaline (NA) plays an important role in modulating processes involved in learning and memory. The present study investigated the effect of NA depletion on stress-induced changes on memory performance in the mouse. Central NA depletion was induced using the selective neurotoxin N-(2-chloroethyl)- N-ethyl-2 bromobenzylamine (DSP-4) and verified by high performance liquid chromatography (HPLC). A novel cage stress procedure involving exposure to a new clean cage for 1 h per day, 4 days per week for 4 weeks, was used to produce stress-induced memory deficits measured using the object recognition task. 50 mg/kg DSP-4 produced large and sustained reductions in NA levels in the frontal cortex and hippocampus measured 24 h, 1 week and 5 weeks after treatment. Four weeks of exposure to novel cage stress induced a memory deficit in the object recognition task which was prevented by DSP-4 pre-treatment (50 mg/kg 1 week before the commencement of stress).These findings indicate that chronic environmental stress adversely affects recognition memory and that this effect is, in part, mediated by the noradrenergic stress response. The implication of these findings is that drugs targeting the noradrenergic system to reduce over-activity may be beneficial in the treatment of stress-related mental disorders such as post-traumatic stress disorder or anxiety in which memory is affected.

Amphetamine-induced locomotion, behavioral sensitization to amphetamine, and striatal D 2 receptor function in rats with high or low spontaneous exploratory activity: Differences in the role of locus coeruleus

Individual differences in novelty-related behavior are associated with sensitivity to various neurochemical manipulations. In the present study the amphetamine-induced locomotor activity and behavioral sensitization to amphetamine (0.5 mg/kg) was investigated in rats with high or low spontaneous exploratory activity (HE- and LE-rats, respectively) after partial denervation of the locus coeruleus (LC) projections with a low dose of the selective neurotoxin DSP-4 ( N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine; 10 mg/kg). DSP-4 produced a partial depletion (about 30%) of noradrenaline in the frontal cortex of both HE- and LE-rats; additionally the levels of metabolites of dopamine and 5-HT were reduced in the frontal cortex and nucleus accumbens of the LE-rats. Amphetamine-stimulated locomotor activity was attenuated by the DSP-4 pretreatment only in the HE-rats and this effect persisted over repeated testing. Behavioral sensitization to repeated amphetamine was evident only in the LE-rats with intact LC projections. Repeated amphetamine treatment reduced D 2 receptor mediated stimulation of [ 35S]GTPγS-binding and dopamine-dependent change in GDP-binding affinity in the striatum, but only in HE-rats. The absence of amphetamine sensitization in HE-rats could thus be related to the downregulation by amphetamine of the G protein stimulation through D 2 receptors. Conclusively, acute and sensitized effects of amphetamine depend on the integrity of LC projections but are differently regulated in animals with high or low trait of exploratory activity. These findings have implications to the neurobiology of depression, drug addiction, and attention deficit hyperactivity disorder.

Effects of low dose N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine administration on exploratory and amphetamine-induced behavior and dopamine D 2 receptor function in rats with high or low exploratory activity

Individual differences in behavioral traits are associated with sensitivity to various neurochemical and psychopharmacological manipulations. In this study exploratory and amphetamine-induced behavior in rats with persistently high or low exploratory activity (HE and LE, respectively) was examined before and after a partial denervation of the locus coeruleus (LC) projections with the selective neurotoxin DSP-4 ( N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine; 10 mg/kg). Partial LC denervation prevented the increase in exploratory activity over repeated test sessions in the LE animals, but had no effect in HE-rats. Amphetamine- (0.5 mg/kg) induced locomotor activity was attenuated by DSP-4 pretreatment only in HE-rats. These results suggest differential involvement of LC noradrenergic transmission in novelty- and amphetamine-induced behavior in animals with persistent differences in novelty-related behavior. In addition to partial noradrenaline depletion in the frontal cortex and hippocampus, which occurred in both HE- and LE-rats, DSP-4 treatment also decreased the content of dopamine and its metabolites in the nucleus accumbens, and the metabolite levels in striatum, but only in the LE-animals. 5-HIAA levels were also reduced in the nucleus accumbens and striatum in LE-rats by the neurotoxin. D 2 receptor function, as determined by dopamine-stimulated [ 35S]GTPγS binding, was increased by DSP-4 treatment in the striatum of LE-rats, but reduced in HE-rats. No effect of partial LC denervation was found on dopamine-stimulated [ 35S]GTPγS binding in the nucleus accumbens. Together these findings suggest that LC noradrenergic neurotransmission is differently involved in dopaminergic mechanisms which mediate novelty-related vs amphetamine-induced behavior.

Interaction of stress and noradrenergic drugs in the control of picrotoxin-induced seizures

To evaluate the possible role of noradrenergic system in the anticonvulsant effect of swim stress, the mice were prior to exposure to swim stress and the i.v. infusion of picrotoxin, pre-treated with desipramine (a noradrenaline reuptake inhibitor), N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine (DSP-4, a neurotoxin which destructs noradrenergic axons) or alpha-methyl- p-tyrosine (α-MPT, an inhibitor of catecholamine synthesis) and the latency to the onset of two convulsant signs and death was registered. While in control unstressed animals desipramine (20 mg/kg i.p.) and alpha-MPT (200 mg/kg i.p.) failed to affect, DSP-4 (50 mg/kg i.p., given 3 weeks prior to experiment) tended to decrease the dose of picrotoxin needed to produce tonic hindlimb extension (THE) and death. Swim stress prolonged the latency, i.e. increased (64–116% above control) the dose of picrotoxin needed to produce convulsant signs and death. In swim stressed mice desipramine enhanced the doses of picrotoxin needed to produce running–bouncing clonus (RB clonus), THE and death. α-MPT and DSP-4 pre-treatment failed to prevent the anticonvulsant effect of stress. Moreover, the effect of stress was greater in DSP-4 pre-treated mice. Although further studies are needed, the results suggest that the integrity of noradrenergic system is not substantial for the anticonvulsant effect of stress.

Control of 5-Hydroxytryptamine Release in the Dorsal Raphe Nucleus by the Noradrenergic System in Rat Brain. Role of α-Adrenoceptors

The interactions between the brainstem serotonergic (5-hydroxytryptamine, 5-HT) and noradrenergic (NA) systems are important for the pathophysiology and treatment of affective disorders. We examined the influence of alpha-adrenoceptors on 5-HT and NA release in the rat dorsal raphe nucleus (DR) using microdialysis. 5-HT and NA concentrations in DR dialysates were virtually suppressed by TTX and increased by veratridine. The local and systemic administration of the alpha(1)-adrenoceptor antagonist prazosin reduced the DR 5-HT output but not that of NA. The maximal 5-HT reduction induced by local prazosin administration (-78% at 100 microM) was more marked than by its systemic administration (-43% at 0.3 mg/kg). The local application of NA and desipramine, to increase the tone on DR alpha(1)-adrenoceptors, did not enhance 5-HT release. The local (100 microM) or systemic (0.1-1 mg/kg s.c.) administration of clonidine reduced 5-HT and NA release (-48 and -79%, respectively, at 1 mg/kg), an effect reversed by RX-821002, which by itself increased both amines when given systemically. DSP-4 pretreatment prevented the effects of clonidine on 5-HT, suggesting the participation of alpha(2)-adrenoceptors on NA elements. Moreover, the systemic effect of clonidine on 5-HT (but not NA) was cancelled by lesion of the lateral habenula and by anesthesia, and was slightly enhanced by cortical transection. These data support the view that alpha(1)-adrenoceptors in the DR tonically stimulate 5-HT release, possibly at nearly maximal tone. Likewise, the 5-HT release is modulated by alpha(2)-adrenoceptors in NA neurons and in forebrain areas involved in the distal control of 5-HT neurons.

Noradrenergic lesions differentially alter the antidepressant-like effects of reboxetine in a modified forced swim test

The novel antidepressant reboxetine is a selective norepinephrine reuptake inhibitor. In this study, the antidepressant-like effects of reboxetine were characterized in a modified rat forced swim test. Further, in order to investigate the role of the locus coeruleus and lateral tegmental noradrenergic systems in the mediation of reboxetine's effects, the impact of different chemical lesions of these two pathways was examined on the behavioral responses induced by reboxetine in the forced swim test. Reboxetine (5–20 mg/kg, s.c.) dose-dependently decreased immobility and swimming behavior in the forced swim test while it simultaneously increased climbing behavior. These effects were similar to those previously demonstrated with tricyclic antidepressants and are indicative of reboxetine's effects on the noradrenergic system. Discrete local injections of the neurotoxin 6-hydroxydopamine were employed to lesion the ventral noradrenergic bundle arising from cells located in the lateral tegmentum. This resulting lesion completely prevented reboxetine (10 mg/kg, s.c.)-induced decreases in immobility and increases in climbing behavior, demonstrating that an intact ventral noradrenergic bundle is required for the manifestation of reboxetine-induced antidepressant-like behavior in the test. In contrast, lesions of the dorsal noradrenergic bundle which consists of neurons arising from the nucleus locus coereleus, were achieved by systemic pretreatment with the selective noradrenergic neurotoxin N-(2-chloroethyl)- N-2-bromobenzylamine (DSP-4; 50 mg/kg, i.p.). The ability of reboxetine (10 mg/kg, s.c.) to increase climbing and decrease immobility was augmented by DSP-4 pretreatment. Furthermore, neither lesions of the dorsal noradrenergic bundle nor the ventral noradrenergic bundle altered baseline immobility scores in the forced swim test. Taken together, these data suggest that forebrain regions innervated by these two distinct noradrenergic pathways exert opposing influences on the behavioral response to reboxetine in the rat forced swim test.

Effects of alpha-adrenoceptor agonists in chronic morphine administered DSP4-treated rats: evidence for functional cross-sensitization.

Five experiments were performed to study the effects of the Alpha-adrenoceptor agonists, clonidine and guanfacine, upon spontaneous motor activity in chronically morphine administered DSP4-treated and control rats. DSP4 (2 x 50 mg/kg, with a 10-day interval between injections) and vehicle (distilled water) were injected i.p., on each occasion 30 min after zimeldine (20 mg/kg). Morphine dosages were raised incrementally from 5 mg/kg (Days 1-3), through 10 mg/kg (Days 4-7) and 20 mg/kg (Days 8-14), to 30 mg/kg (Days 15-20). Motor activity testing occurred on Day 21, Day 22 as well as in Experiments II-V, (from 1st morphine injection). DSP4 pretreatment and chronic morphine injections each reduced motor activity during the first 30 min of testing; combined DSP4 and morphine treatment potentiated the hypoactivity. Habituation quotients indicated deficits in habituation to the novel test environment by the Vehicle-morphine (Quoteint2 only) and DSP4-morphine groups. Acute clonidine treatment (0.04 mg/kg s.c.) reduced motor activity during the first 30 min of testing but attenuated or blocked the morphine-induced hypoactivity in DSP4-treated and control rats. During the 60-90 min test period, clonidine, but not guanfacine (0.08 mg/kg), potentiated morphine-induced hyperactivity in control rats; acute clonidine enhanced this effect, whereas acute guanfacine reduced it, in the DSP4-treated rats. The enhanced hyperactivity of morphine-clonidine suggest a cross-sensitivity effect. Naloxone (0.1 mg/kg s.c.), injected after the 1st 30-min of testing, potentiated markely the clonidine-induced elevations of motor activity in morphine-administered control rats; in the DSP4-treated rats, these effects were dramatically potentiated, underlining the cross-sensitivity effect. Acute guanfacine treatment reduced motor activity during the first 30 min of testing but did not attenuate reliably morphine-induced hypoactivity in control or DSP4 rats. Naloxone did not potentiate the guanfacine-induced hyperactivity of morphine-administered control rats but induced a marked enhancement in the DSP4-treated rats, a specific case of cross-reactivity. The major findings pertain to a cross-sensitization effect of morphine upon clonidine-induced motor activity in both DSP4-treated and control rats, and to a lesser extent between morphine and guanfacine in NA-denervated rats only. The results may offer interactive implications for noradrenergic-opiate system functioning that may be of influence under neuropathological conditions.

Neuropeptide Y attenuates the effect of locus coeruleus denervation by DSP-4 treatment on social behaviour in the rat

Noradrenaline (NA) has been implicated in both increase and reduction of anxiety. Selective destruction of nerve endings of the locus coeruleus projections by DSP-4 has been shown to reduce active behaviour in novel situations by enhancing anxiety. In the present study, DSP-4 (50 mg/kg) treatment reduced locomotor activity and time spent in social interaction in rats placed into a novel environment together with an unfamiliar rat, indicating an anxiogenic-like effect. The effect of DSP-4 on time spent in social interaction was completely antagonized by intracerebroventricular administration of neuropeptide Y (NPY) (1 μg) which had no effect of its own on this measure. The present study thus supports the idea that DSP-4 pretreatment is anxiogenic in novel situations and suggests a functional relationship of NA- and NPY-using neural mechanisms in the regulation of social behaviour.

Effects of clonidine and alpha-adrenoceptor antagonists on motor activity in DSP4-treated mice II: interactions with apomorphine.

Adult mice were administered either the noradrenaline (NA) neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) or distilled water (control), 10-12 days before motor activity testing, and 6 h before testing all the mice were administered reserpine (10 mg/kg), the monoamine-depleting agent. The interactive effects of (I) clonidine, the alpha(2)-adrenoceptor agonist, with the dopamine (DA) agonist, apomorphine, and the alpha(2)-antagonist, yohimbine, and (II) with either yohimbine or the alpha(1)-antagonist, prazosin, upon motor behaviour in activity test chambers were studied in reserpinized DSP4-treated and control mice. It was shown that apomorphine (3 mg/kg) increased locomotor and total activity in both reserpinized DSP4-treated and control mice but the effect was attenuated in the DSP4 mice. Co-administration of clonidine (3 mg/kg) with apomorphine potentiated the effects of apomorphine on motor activity and this effect was enhanced markedly by DSP4 pretreatment. Yohimbine (10 mg/kg) antagonized the motor activity-stimulating effects of apomorphine in both DSP4-treated and control mice. Co-administration of clonidine with apomorphine, following yohimbine, restored motor activity levels to those obtained in the absence of yohimbine and this effect upon locomotor activity was enhanced by DSP4 pretreatment. The effects of clonidine on motor activity were enhanced by NA-denervation. Prazzosin (3 mg/kg) enhanced the locomotor activity of both reserpinized DSP4-treated and control mice after the initial 30-min period but was not affected by DSP4 treatment. Analysis of post-decapitation convulsions (PDCs) indicated loss of the reflex by DSP4 pretreatment. Reserpine pretreatment abolished the initial, exploratory phase (30 min) of motor activity. These results demonstrate interactions between NA and DA systems that may bear eventual relevance to neurologic disorders such as parkinsonism.

Modulatory role of 5-HT 3 receptors in mediation of apomorphine-induced aggressive behaviour in male rats

We have studied the effects of serotonin (5-HT) 5-HT 3 receptor agonists 1-phenylbiguanide (1-PBG) and 1-(m-chlorophenyl)-biguanide (mCPBG), and antagonists 3-tropanyl-3,5-dichlorobenzoate (MDL-72222) and tropisetron (3-tropanyl-indole-3-carboxylate HCl; ICS-205930) on apomorphine-induced aggressive behaviour in normal or DSP-4 [ N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine hydrochloride] pre-treated male Wistar rats. DSP-4 (50 mg/kg) pre-treatment significantly accelerated the development of apomorphine-induced aggressive behaviour. mCPBG (1.0 and 10 mg/kg) did not modify the aggressiveness, but 1-PBG (3.0 and 30 mg/kg) attenuated the aggressiveness in normal but not DSP-4 pre-treated rats. MDL-72222 (0.4 and 4.0 mg/kg) attenuated the aggressive behaviour in normal rats, tropisetron (0.3 mg/kg) had an antiaggressive effect only by citalopram (10 mg/kg) challenge. MDL-72222 and tropisetron were ineffective in DSP-4 pre-treated rats. In conclusion, our results indicate that the 5-HT 3 receptors modulate the apomorphine-induced aggressive behaviour and the 5-HT 3 receptor antagonists have moderate antiaggressive effect in this test.

Depletion of brain norepinephrine: differential influence on anxiolyic treatment effects.

Previous studies have demonstrated that anxiolytic-like anticonflict effects can be produced by either (1) acute administration of traditional anti-anxiety compounds (benzodiazepines or barbiturates) or (2) chronic administration of tricyclic (TCA) or monoamine oxidase inhibitor (MAOI) anti-depressants. The present study determined the effects of noradrenergic neuronal depletion on these distinct anticonflict treatments. After 3 weeks of training in a repeated measures drink suppression conflict paradigm, water-restricted rats consumed 11-14 ml water/session (unpunished responding) and accepted 25-40 shocks/session (punished responding) during control (i.e., non-drug) 10-min test sessions. The noradrenergic neurotoxin DSP4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride; 65 mg/kg, IP] or its vehicle (saline; 1 ml/kg) was administered after 3 weeks of conflict testing. Conflict behavior was then evaluated for 8 weeks post-treatment. In separate groups of DSP4- and vehicle-pretreated subjects, the effects of acute administration (10-min pretreatment) of phenobarbital (5-40 mg/kg) or alprazolam (0.3-2.5 mg/kg) were determined. In a third experiment, the effects of chronic treatment with the TCA desipramine (DMI; 5 mg/kg, twice daily for 8 weeks) or the non-selective MAOI phenelzine (4.0 mg/kg, twice daily for 8 weeks) on conflict behavior were determined in additional groups of DSP4- or vehicle-pretreated subjects. DSP4 treatment produced a modest yet statistically significant decrease in punished responding (i.e., anxiogenic-like effect) relative to vehicle controls. DSP4 pretreatment did not alter the anticonflict effects of acute alprazolam or phenobarbital treatment. In contrast, DSP4 treatment completely abolished the anticonflict effects produced by chronic DMI or chronic phenelzine treatment. Thus, noradrenergic neuronal integrity appears to be required for the anxiolytic-like effects of chronic antidepressant treatment, but not for the anxiolytic-like effects of acute treatment with barbiturates and benzodiazepines.