Altered activity of the locus coeruleus in an animal model of depression

In rats showing depression of active behavior, locus coeruleus (LC) neurons were not inhibited as they are normally; in particular, electrophysiologic recording showed LC neurons were hyperresponsive to excitatory input. Moreover, the degree to which LC neurons of individual animals were hyperresponsive correlated positively with the degree to which active behavior was depressed. Recently, hyperresponsiveness of LC neurons has been found to occur when the inhibitory influence of alpha 2-receptors on LC firing is blocked. Pharmacologic blockade of alpha 2-receptors in animals showing behavioral depression was not able to increase responsiveness of LC neurons as in normal animals, indicating that alpha 2-receptors were subnormally activated in behavioral depression. These results show that activity of the principle noradrenergic cell group in the brain, the locus coeruleus, is altered in behavioral depression in an animal model, and suggest that abnormalities in this system and its regulatory elements, such as alpha 2-receptors, may be present in some types of clinical depression.     

Evidence for the locus coeruleus involvement in desipramine action in animal models of depression

Desipramine (DMI) effectively antagonized hypothermia induced by reserpine and clonidine in rats. DMI effects were attenuated or even abolished after electrolytic or 6-hydroxydopamine-induced lesion of the locus coeruleus (LC) as well as by administration of DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine), a selective noradrenergic neurotoxin. Contrary to the LC lesions, electrolytic destruction of the ventral noradrenergic bundle did not change DMI action but antagonized reserpine-induced hypothermia by itself. Our results underline a possible involvement of the LC system in mechanism of antidepressive action, which was suggested previously in this laboratory.     

Galanin decreases the activity of locus coeruleus neurons in vitro

A brain slice preparation was used to examine the effects of galanin on the spontaneous firing rate of locus coeruleus noradrenergic neurons. Galanin (10(-9)-10(-7) M), added to the bath, inhibited the firing of 14 out of 19 neurons in a concentration-dependent manner. The observed effect was quite variable, ranging from 20 to 100% at 10(-7) M. Experiments performed in low-Ca2+, high-Mg2+ medium also showed a significant inhibition by galanin (10(-7) M) in three out of five neurons, which suggests that the peptide acts directly.     

Convergent regulation of locus coeruleus activity as an adaptive response to stress

Although hypothalamic-pituitary-adrenal axis activation is generally considered to be the hallmark of the stress response, many of the same stimuli that initiate this response also activate the locus coeruleus-norepinephrine system. Given its functional attributes, the parallel engagement of the locus coeruleus-norepinephrine system with the hypothalamic-pituitary-adrenal axis serves to coordinate endocrine and cognitive limbs of the stress response. The elucidation of stress-related afferents to the locus coeruleus and the electrophysiological characterization of these inputs are revealing how the activity of this system is fine-tuned by stressors to facilitate adaptive cognitive responses. Emerging from these studies, is a picture of complex interactions between the stress-related neuropeptide, corticotropin-releasing factor (CRF), endogenous opioids and the excitatory amino acid neurotransmitter, glutamate. The net effect of these interactions is to adjust the activity and reactivity of the locus coeruleus-norepinephrine system such that state of arousal and processing of sensory stimuli are modified to facilitate adaptive behavioral responses to stressors. This review begins with an introduction to the basic anatomical and physiological characteristics of locus coeruleus neurons. The concept that locus coeruleus neurons operate through two activity modes, i.e., tonic vs. phasic, that determine distinct behavioral strategies is emphasized in light of its relevance to stress. Anatomical and physiological evidence are then presented suggesting that interactions between stress-related neurotransmitters that converge on locus coeruleus neurons regulate shifts between these modes of discharge in response to the challenge of a stressor. This review focuses specifically on the locus coeruleus because it is the major source of norepinephrine to the forebrain and has been implicated in behavioral and cognitive aspects of stress responses.     

Morphine increases locus coeruleus noradrenergic neuronal activity in vitro

Morphine produced a dose-dependent increase in the activity of norepinephrine-containing locus coeruleus neurons recorded from mouse brain slices in vitro. The response was not changed in a low calcium-high magnesium incubation medium, indicating that the observed effects are the result of a direct action of morphine on locus coeruleus neurons. When the mice were anesthetized with chloral hydrate prior to preparation of tissue slices, morphine produced a dose-dependent suppression of noradrenergic neuronal activity. These studies suggest that previous data showing a decrease in activity of locus coeruleus neurons following incubation with morphine in vitro are due to the use of anesthesia.     

Buspirone increases locus coeruleus noradrenergic neuronal activity in vitro

Buspirone, a non-benzodiazepine anxiolytic agent, produced dose-dependent increases in the activity of norepinephrine-containing locus coeruleus neurons recorded from mouse brain slices in vitro. The response was not changed in a low calcium/high magnesium incubation medium, indicating that the observed effects were the result of a direct action of buspirone on locus coeruleus neurons. These data suggest that noradrenergic neurons may not be as important in mediating anxiety states as previously suggested.     

Dependence of serotonin release in the locus coeruleus on dorsal raphe neuronal activity

The serotonergic innervation of the locus coeruleus paetly derives from the dorsal raphe nucleus (DRN). Using the push-pull superfusion technique, we investigated whether and to what extent the release of serotonin and the extracellular concentration of its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the locus coeruleus are influenced by the neuronal activity of the DRN. In anaesthetized rats, a push-pull cannula was inserted into the locus coeruleus, which was continuously superfused with artificial cerebrospinal fluid (aCSF). Serotonin and 5-HIAA levels in the superfusate were determined by HPLC combined with electrochemical detection. Electrical stimulation (5 Hz, 300 μA, 1 ms) of the DRN for 5 min, or its chemical stimulation by microinjection of glutamate (3.5 nmol, 50 nl), led to an increased release of serotonin in the locus coeruleus and to a slight (2 mmHg) decrease in blood pressure. Superfusion of the locus coeruleus with tetrodotoxin (1 μM) abolished the increase in the release rate of serotonin evoked by electrical stimulation of the DRN, while the slight fall in blood pressure was not influenced. Thermic lesion (75 °C, 1 min) of the DRN elicited a pronounced decline in serotonin release rate within the locus coeruleus, the maximum decrease being 52%. The decrease in the release of serotonin was associated with a long-lasting rise in blood pressure. Microinjection of the serotonin neurotoxin 5,7-dihydroxytryptamine (5 μg, 250 nl) into the DRN led to an initial increase in the serotonin release rate that coincided with a short-lasting fall in blood pressure. Subsequently, the release of serotonin was permanently reduced and was associated with hypertension. Microinjection of the 5-HT_1A receptor agonist (±)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT; 7.5 nmol, 50 nl) into the DRN led to a long-lasting reduction of the release rate of serotonin in the locus coeruleus. Microinjection of 8-OH-DPAT into the DRN also slightly lowered blood pressure (3 mmHg). Neither stimulations nor lesion of the DRN, nor microinjection of 8-OH-DPAT into this raphe nucleus, altered the extracellular concentration of 5-HIAA. Judging from the present biochemical results it appears that the serotonergic afferents to the locus coeruleus originate to more than 50% from cell bodies located in the DRN. The neuronal serotonin release in the locus coeruleus is modulated by 5-HT_1A receptors lying within the DRN. Changes in blood pressure and release of serotonin elicited by stimulating or lesioning the DRN point to the importance of serotonergic neurons extending between this raphe nucleus and the locus coeruleus in central cardiovascular control. Received: 5 November 1998 / Accepted: 21 February 1999     

Postsynaptic serotonergic blockade following chronic antidepressive treatment with trazodone in an animal model of depression

Acute pretreatment with clinically equivalent doses of antidepressive drugs has been observed to block D,L-5-hydroxytryptophan (5-HTP) induced behavioral depression in rats working on a food-reinforced operant schedule. Data from studies designed to distinguish presynaptic from postsynaptic events, indicated that the antidepressants were acting in part as blockers of postsynaptic serotonergic receptors. Using the same 5-HTP model of depression, we studied both the chronic and acute effects of a recently introduced antidepressant, triazolopyridine compound. Rats working for milk reinforcement and exhibiting behavioral depression following administration of 50 mg/kg 5-HTP were pretreated (one hr before 5-HTP) with 1, 2, or 4 mg/kg trazodone with resulting blockade of 5-HTP induced depression of 35, 62 and 70% respectively. Chronic administration of trazodone (2 mg/kg trazodone/day) also resulted in a significant blockade of the 5-HTP effect (75%). Neither 2 mg/kg or 4 mg/kg trazodone was found to potentiate the shorter period of depression following 25 mg/kg 5-HTP. Chronic treatment with the antidepressant drugs, amitriptyline or mianserin also blocked 5-HTP depression. Thus, as in our earlier studies, these data suggest an important postsynaptic mechanism associated with chronic administration of trazodone, amitriptyline and mianserin which could be implicated in the therapeutic effectiveness of these drugs. The potency of trazodone in relation to other antidepressant drugs in our behavioral model of depression paralleled their potency in displacing radioligand binding to 5-HT receptors, and gives additional support for the new hypersensitive postsynaptic serotonin receptor theory of depression.     

Modulation of the firing activity of noradrenergic neurones in the rat locus coeruleus by the 5-hydroxtryptamine system

1. The aim of the present study was to investigate the putative modulation of locus coeruleus (LC) noradrenergic (NA) neurones by the 5-hydroxytryptaminergic (5-HT) system by use of in vivo extracellular unitary recordings and microiontophoresis in anaesthetized rats. To this end, the potent and selective 5-HT_1A receptor antagonist WAY 100635 (N-{2-[4(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide trihydroxychloride) was used.
2. In the dorsal hippocampus, both local (by microiontophoresis, 20 nA) and systemic (100 μg kg⁻¹, i.v.) administration of WAY 100635 antagonized the suppressant effect of microiontophorectically-applied 5-HT on the firing activity of CA₃ pyramidal neurones, indicating its antagonistic effect on postsynaptic 5-HT_1A receptors.
3. WAY 100635 and 5-HT failed to modify the spontaneous firing activity of LC NA neurones when applied by microiontophoresis. However, the intravenous injection of WAY 100635 (100 μg kg⁻¹) readily suppressed the spontaneous firing activity of LC NA neurones.
4. The lesion of 5-HT neurones with the neurotoxin 5,7-dihydroxytryptamine increased the spontaneous firing activity of LC NA neurones and abolished the suppressant effect of WAY 100635 on the firing activity of LC NA neurones.
5. In order to determine the nature of the 5-HT receptor subtypes mediating the suppressant effect of WAY 100635 on NA neurone firing activity, several 5-HT receptor antagonists were used. The selective 5-HT₃ receptor antagonist BRL 46470A (10 and 100 μg kg⁻¹, i.v.), the 5-HT_1D receptor antagonist GR 127935 (100 μg kg⁻¹, i.v.) and the 5-HT_1A/1B receptor antagonist (−)-pindolol (15 mg kg⁻¹, i.p.) did not prevent the suppressant effect of WAY 100635 on the firing activity of LC NA neurones. However, the suppressant effect of WAY 100635 was prevented by the non-selective 5-HT receptor antagonists spiperone (1 mg kg⁻¹, i.v.) and metergoline (1 mg kg⁻¹, i.v.), by the 5-HT₂ receptor antagonist ritanserin (500 μg kg⁻¹, i.v.). It was also prevented by the 5-HT_1A receptor/α_1D-adrenoceptor antagonist BMY 7378 (1 mg kg⁻¹, i.v.) and by the α₁-adrenoceptor antagonist prazosin (100 μg kg⁻¹, i.v.).
6. These data support the notion that the 5-HT system tonically modulates NA neurotransmission since the lesion of 5-HT neurones enhanced the LC NA neurones firing activity and the suppressant effect of WAY 100635 on the firing activity of NA neurones was abolished by this lesion. However, the location of the 5-HT_1A receptors involved in this complex circuitry remains to be elucidated. It is concluded that the suppressant effect of WAY 100635 on the firing activity of LC NA neurones is due to an enhancement of the function of 5-HT neurones via a presynaptic 5-HT_1A receptor. In contrast, the postsynaptic 5-HT receptor mediating this effect of WAY 100635 on NA neurones appears to be of the 5-HT_2A subtype.

     

Hippocampus and locus coeruleus activity on rats chronically treated with diazepam

The neural mechanisms underlying benzodiazepine (BZD) dependence remain equivocal. The present studies tested the hypothesis that similar neural circuitry might be involved in the effects of chronic 7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepine-2(1H)-one, diazepam (DZ, Roche), administration and withdrawal. The results of our study showed an increased hippocampal synaptic plasticity in slices from rats chronically treated with DZ (5 mg/kg/18 days), assessed as a decrease of the threshold in the stimulation rate for long-term potentiation (LTP) elicitation. Rats with the same schedule of DZ administration but without signs of withdrawal behaved similarly to vehicle-treated ones (VEH), in the threshold to induce LTP. Furthermore, the activity of locus coeruleus (LC) norepinephrine (NE) neurons in rats tested 24 h after the last DZ injection showed a significant increase. On the other hand, rats that after chronic DZ administration did not develop signs of withdrawal and exhibited a similar pattern of discharge on LC-NE nucleus compared with their controls. We conclude that chronic DZ administration enhances both hippocampal synaptic plasticity and activity of LC-NE neurons. This neural system could be the biological substrate underlying the behavioral alterations accompanying chronic DZ administration and withdrawal.     

Antitumor activity of trovafloxacin in an animal model

Quinolone antibiotics come close to being ideal chemotherapeutic agents in that they are administered orally, are concentrated in cells and tissue, are readily available, relatively safe and exhibit increased activity against both bacteria and tumor cells both in vitro and in vivo. Our objective was to evaluate the in vivo activity of trovafloxacin and ciprofloxacin against murine leukemic cells in neutropenic mice with lung infection due to Klebsiella pneumoniae. The results showed that both trovafloxacin and ciprofloxacin were effective in clearing lung infection. However, trovafloxacin, but not ciprofloxacin, was effective in preventing metastasis of leukemia cells to the lungs and other tissue and in prolonging the survival of mice.     

Differential effects of an anxiogenic beta-carboline on single unit activity in the locus coeruleus and substantia nigra of the rat brain

The firing rates of single units in the substantia nigra pars reticulata, substantia nigra pars compacta and the locus coeruleus were recorded during the intravenous administration of beta-carboline-3-carboxylic acid ethyl ester (beta CCE). beta-Carboline-3-carboxylic acid produced a dose-dependent excitation in all units tested in the substantia nigra pars reticulata and a small inhibitory effect on some units in the substantia nigra pars compacta. It had no effect on the firing rates of single units in the locus coeruleus, but did prove effective in reversing inhibition induced by diazepam in this nucleus. The relevance of the findings to the anxiogenic effects of beta-carboline-carboxylic acid ethyl ester are discussed.     

Chronic morphine treatment increases cyclic AMP-dependent protein kinase activity in the rat locus coeruleus

We have studied a possible role for cyclic AMP-dependent protein kinase in mediating opiate addiction in the central nervous system by focusing on the rat locus coeruleus. This brain region is well suited for these studies because it is relatively homogeneous and because a wealth of electrophysiological and behavioral data indicate that it plays an important role in mediating the chronic effects of opiates in animals, including humans. It was found that chronic, but not acute, in vivo treatment of rats with morphine increased cyclic AMP-dependent protein kinase activity in the locus coeruleus with a time course that closely paralleled the time course by which locus coeruleus neurons become tolerant to and dependent on opiates, based on electrophysiological studies. Concomitant administration of the opiate receptor antagonist naltrexone was found to block the effect of chronic morphine treatment on protein kinase activity, indicating that the effect is mediated via specific activation of opiate receptors. In contrast, chronic morphine treatment did not alter protein kinase activity in several other brain regions studied, including the neostriatum, frontal cortex, and dorsal raphe. We propose that the observed increase in cyclic AMP-dependent protein kinase activity in the locus coeruleus contributes to the biochemical basis of opiate addiction.     

Opiate-receptor interactions on single locus coeruleus neurones

Intracellular recordings were made from neurones of the rat locus coeruleus (LC) which were located in a slice of pons superfused in vitro. Opioid agonists and antagonists were applied by adding them to the superfusing solution; normorphine and enkephalin analogues were also applied by ejecting a few nanoliters of a solution which contained the drugs from a pipette situated above the tissue slice. Opioid agonists hyperpolarized LC neurones. This has been shown previously to result from an increase in the membrane potassium conductance. The lowest concentration of normorphine which was effective was 30 nM, the EC50 was 1 microM, and the maximum effect was observed with 30 microM. The irreversible antagonist beta-funaltrexamine (beta-FNA) was used to estimate the dissociation equilibrium constants; these ranged from 9-16 microM for normorphine and [Met5]enkephalin and was about 2 microM for [D-Ala2,D-Leu5]enkephalin. beta-FNA also blocked the hyperpolarization caused by [D-Ala2,D-Leu5]enkephalin, ethylketacyclazocine, and [D-Ser2,D-Leu5] enkephalin-Thr. Naloxone reversibly antagonized the hyperpolarizations caused by normorphine and [D-Ala2,D-Leu5]enkephalin, with a dissociation equilibrium constant of 2 nM. It is suggested that the opioid hyperpolarization of LC neurones is mediated by a receptor having a high affinity for naloxone, previously termed a mu-receptor. The affinity of this receptor for normorphine appears to be 3 to 4 orders or magnitude lower than its affinity for naloxone.     

No evidence for functional imidazoline receptors on locus coeruleus neurons

₂-Adrenoceptor agonists inhibit the firing of locus coeruleus (LC) neurons. It was recently observed that the ₂-adrenoceptor agonists clonidine, rilmenidine and cirazoline, when injected intravenously in anaestbetized rats pretreated with the irreversible ₂-adrenoceptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), excite the LC. The effect was attributed to activation of 11 imidazoline receptors. The aim of the present experiments was to characterize the direct effect of ₂-adrenoceptor and I₁ imidazoline receptor agonists on LC neurons.Electrical activity of LC neurons was extracellularly recorded in midpontine slices prepared from the rat brain. Concentration-response curves were obtained for the ₂-agonist noradrenaline and the mixed I₁/₂-receptor agonists clonidine, rilmenidine and moxonidine in slices without treatment and in slices treated with 6-chloro-N-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F86466) or EEDQ, ₂-adrenoceptor antagonists with low affinity for I₁ and I₂ imidazoline receptors, respectively. All four agonists concentration-dependently reduced the firing rate of the neurons, with full inhibition at higher concentrations. SK&F86466 shifted the concentration-response curves of the agonists to the right; the calculated antagonist dissociation constants are compatible with an effect of the agonists on ₂-adrenoceptors. EEDQ completely prevented the inhibition by the agonists. Neither in SK&F86466- nor in EEDQ-treated slices was an excitation by clonidine, rilmenidine and moxonidine observed.We conclude that the LC neurons do not possess functional I₁ (and also no I₂) imidazoline receptors. The effects of noradrenaline, clonidine, rilmenidine and moxonidine on the neurons can be fully explained with an interaction with inhibitory ₂-adrenoceptors (probably of the _2D subtype). The excitation of the LC by imidazoline receptor agonists under in vivo conditions, hence, is not a direct effect on the neurons of the LC.     

Cyclobenzaprine effects on locus coeruleus cells in tissue slice

Tissue slices 400 μ thick were taken from the brain stem, at the level of the locus coeruleus, of 150–250 g Sprague-Dawley rats. Microe1ectrodes were placed in the locus coeruleus under visual control, and a cell whose discharge rate would decrease with microiontophoretic application of norepinephrine or clonidine was sought. Cyclobenzaprine (CBZ) was then introduced into the perfusion medium at a concentration equivalent to 1 mg/kg body weight of the whole animal. Discharge rates before and during CBZ administration were compared. The six cells with initial discharge rates between 2 and 10 Hz decreased firing with CBZ, whereas the four cells with initial rates between 0.5 and 1.5 Hz increased their rates with CBZ.     

The proconvulsive activity of quinolone antibiotics in an animal model

The side-effect profile of quinolone antibiotics in man includes CNS disturbances such as dizziness, insomnia and convulsions. Although it has been suggested that the proconvulsive liability of quinolones involves an interaction with GABA receptors in the central nervous system, no animal model has been described to evaluate or confirm the mechanism of this effect. The proconvulsive activity of the quinolone antibiotics, nalidixic (NAL) and oxolinic (OXO) acid were tested in male mice following oral doses of 10-100 mg/kg utilizing the convulsive stimuli pentylenetetrazole (PTZ), picrotoxin, strychnine or electroshock. While NAL and OXO did not alter the threshold for convulsions induced by PTZ, strychnine or picrotoxin, both agents lowered the threshold for electroshock-induced seizures. Furthermore, the proconvulsive actions of NAL and OXO were completely blocked by the excitatory amino acid receptor antagonists, MK-801 and 2-amino-4-phosphonobutyric acid (AP-4). These data indicate that the mechanism of convulsive liability of quinolone antibiotics does not involve GABA receptor interactions as previously thought, but appears to involve activation of excitatory amino acid (EAA) receptors, possibly located in the optic region of the central nervous system.     

Antidepressant drugs with differing pharmacological actions decrease activity of locus coeruleus neurons

Previous studies suggest that all effective antidepressant (AD) drugs decrease activity of locus coeruleus (LC) neurons. However, little data exist regarding blood levels of drug in these studies, and what data do exist suggest blood levels might have been very high. To assess whether decreased LC activity is produced by drugs that selectively block reuptake for either norepinephrine or serotonin at therapeutically relevant blood levels, effects of chronic administration of desipramine, paroxetine, and escitalopram on LC activity were measured across a range of doses and blood levels of drug. Further, effects of a range of doses of mirtazapine were examined; in that mirtazapine blocks alpha2 adrenergic receptors, it might be anticipated to increase rather than decrease LC activity. Finally, to begin to assess whether the response of LC to ADs was specific to these drugs, effects of four non-AD drugs (single dose) were measured. Drugs were administered via osmotic minipump for 14 d. Electrophysiological recording of LC activity (assessment of both spontaneous firing rate and sensory-evoked 'burst' firing) then took place under isoflurane anaesthesia on the last day of drug treatment. The blood level of drugs present at the end of the recording session was also measured. All AD drugs tested decreased LC spontaneous and sensory-evoked 'burst' firing, and this was observed across a wide range of blood levels for the drugs. Non-AD drugs did not decrease LC activity. The findings of this investigation continue to support the possibility that all effective AD drugs decrease LC activity.