Locus Coeruleus Firing Rate Research Articles

Transcriptomic and proteomic characterization of the locus coeruleus in a rat model of Alzheimer’s disease

AbstractBackgroundThe noradrenergic locus coeruleus (LC) is the first region to accumulate hyperphosphorylated tau pathology (p‐tau) in Alzheimer’s disease (AD). Using TgF344‐AD transgenic rats, which develop endogenous p‐tau in the LC prior to pathology in forebrain regions, we have recently demonstrated age‐dependent effects of p‐tau on LC firing rates. Specifically, LC neurons exhibit early hyperactivity followed by late hypoactivity. These changes in LC firing rates are coincident with the emergence of behavioral phenotypes that characterize different stages of AD (neuropsychiatric symptoms followed by cognitive deficits). Here, we sought to identify molecular mechanisms that could contribute to these changes using RNAscope and proteomics in TgF344‐AD rats.Methods32 male and female TgF344‐AD rats and wild‐type (WT) littermates aged 6 and 15 months were used for this study. Brains were flash‐frozen in isopentane on dry ice and stored at ‐80°C until being sectioned. At the level of the LC, the left hemisphere LC was collected with a 1 mm tissue punch. Punches of the cerebellum were taken as a control to verify LC enrichment in the brainstem punches. The right LC hemisphere was sectioned at 16 µm for processing with fluorescent in situ hybridization using the RNAscope Multiplex Fluorescent V2 Assay according to the manufacturer’s protocol. Probes included noradrenergic markers (tyrosine hydroxylase, norepinephrine transporter) and those that influence LC activity (α2‐adrenergic receptors, GRIA2, GRIN, ADCYAP1R1). Separately, tissue punches were submitted to the Emory Integrated Proteomics Core for label‐free quantification of proteins.ResultsRNAscope revealed no differences in transcript levels of noradrenergic markers at either age. Similarly, there were no differences in transcript levels of α2‐adrenergic receptors, GRIA, or ADCYAP1R1 at either age, or GRIN in 6‐month animals. We observed an increase of GRIN mRNA in 15‐month TgF344‐AD animals, suggesting an upregulation of excitatory neurotransmission.ConclusionsNoradrenergic neuronal identity remains in‐tact up to 15 months of age, a timepoint when there is no LC cell death in these rats. We have also uncovered a potential compensatory mechanism to maintain LC activity in late stages of disease. Proteomics and RNAscope analysis of other markers that may affect LC activity are ongoing.

Subunit-specific expression and function of AMPA receptors in the mouse locus coeruleus.

The locus coeruleus (LC) provides the principal supply of noradrenaline (NA) to the brain, thereby modulating an array of brain functions. The release of NA, and therefore its impact on the brain, is governed by LC neuronal excitability. Glutamatergic axons, from various brain regions, topographically innervate different LC sub-domains and directly alter LC excitability. However, it is currently unclear whether glutamate receptor sub-classes, such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, are divergently expressed throughout the LC. Immunohistochemistry and confocal microscopy were used to identify and localise individual GluA subunits in the mouse LC. Whole-cell patch clamp electrophysiology and subunit-preferring ligands were used to assess their impact on LC spontaneous firing rate (FR). GluA1 immunoreactive clusters were associated with puncta immunoreactive for VGLUT2 on somata, and VGLUT1 on distal dendrites. GluA4 was associated with these synaptic markers only in the distal dendrites. No specific signal was detected for the GluA2-3 subunits. The GluA1/2 receptor agonist (S)-CPW 399 increased LC FR, whilst the GluA1/3 receptor antagonist philanthotoxin-74 decreased it. 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA), a positive allosteric modulator of GluA3/4 receptors, had no significant effect on spontaneous FR. The data suggest distinct AMPA receptor subunits are targeted to different LC afferent inputs and have contrasting effects on spontaneous neuronal excitability. This precise expression profile could be a mechanism for LC neurons to integrate diverse information contained in various glutamate afferents.

Enhanced firing of locus coeruleus neurons and SK channel dysfunction are conserved in distinct models of prodromal Parkinson\u2019s disease

Parkinson’s disease (PD) is clinically defined by the presence of the cardinal motor symptoms, which are associated with a loss of dopaminergic nigrostriatal neurons in the substantia nigra pars compacta (SNpc). While SNpc neurons serve as the prototypical cell-type to study cellular vulnerability in PD, there is an unmet need to extent our efforts to other neurons at risk. The noradrenergic locus coeruleus (LC) represents one of the first brain structures affected in Parkinson’s disease (PD) and plays not only a crucial role for the evolving non-motor symptomatology, but it is also believed to contribute to disease progression by efferent noradrenergic deficiency. Therefore, we sought to characterize the electrophysiological properties of LC neurons in two distinct PD models: (1) in an in vivo mouse model of focal α-synuclein overexpression; and (2) in an in vitro rotenone-induced PD model. Despite the fundamental differences of these two PD models, α-synuclein overexpression as well as rotenone exposure led to an accelerated autonomous pacemaker frequency of LC neurons, accompanied by severe alterations of the afterhyperpolarization amplitude. On the mechanistic side, we suggest that Ca2+-activated K+ (SK) channels are mediators of the increased LC neuronal excitability, as pharmacological activation of these channels is sufficient to prevent increased LC pacemaking and subsequent neuronal loss in the LC following in vitro rotenone exposure. These findings suggest a role of SK channels in PD by linking α-synuclein- and rotenone-induced changes in LC firing rate to SK channel dysfunction.

A Feasibility Study to Investigate Chemogenetic Modulation of the Locus Coeruleus by Means of Single Unit Activity.

AimSelective chemogenetic modulation of locus coeruleus (LC) neurons would allow dedicated investigation of the role of the LC-NA pathway in brain excitability and disorders such as epilepsy. This study investigated the feasibility of an experimental set-up where chemogenetic modification of the brainstem locus coeruleus NA neurons is aimed at and followed by LC unit activity recording in response to clozapine.MethodsThe LC of male Sprague-Dawley rats was injected with 10 nl of adeno-associated viral vector AAV2/7-PRSx8-hM3Dq-mCherry (n = 19, DREADD group) or AAV2/7-PRSx8-eGFP (n = 13, Controls). Three weeks later, LC unit recordings were performed in anesthetized rats. We investigated whether clozapine, a drug known to bind to modified neurons expressing hM3Dq receptors, was able to increase the LC firing rate. Baseline unit activity was recorded followed by subsequent administration of 0.01 and 0.1 mg/kg of clozapine in all rats. hM3Dq-mcherry expression levels were investigated using immunofluorescence staining of brainstem slices at the end of the experiment.ResultsUnit recordings could be performed in 12 rats and in a total of 12 neurons (DREADDs: n = 7, controls: n = 5). Clozapine 0.01 mg/kg did not affect the mean firing rate of recorded LC-neurons; 0.1 mg/kg induced an increased firing rate, irrespective whether neurons were recorded from DREADD or control rats (p = 0.006). Co-labeling of LC neurons and mCherry-tag showed that 20.6 ± 2.3% LC neurons expressed the hM3Dq receptor. Aspecific expression of hM3Dq-mCherry was also observed in non-LC neurons (26.0 ± 4.1%).ConclusionLC unit recording is feasible in an experimental set-up following manipulations for DREADD induction. A relatively low transduction efficiency of the used AAV was found. In view of this finding, the effect of injected clozapine on LC-NA could not be investigated as a reliable outcome parameter for activation of chemogenetically modified LC neurons. The use of AAV2/7, a vector previously applied successfully to target dopaminergic neurons in the substantia nigra, leads to insufficient chemogenetic modification of the LC compared to transduction with AAV2/9.

Parametric characterization of neural activity in the locus coeruleus in response to vagus nerve stimulation

Vagus nerve stimulation (VNS) has emerged as a therapy to treat a wide range of neurological disorders, including epilepsy, depression, stroke, and tinnitus. Activation of neurons in the locus coeruleus (LC) is believed to mediate many of the effects of VNS in the central nervous system. Despite the importance of the LC, there is a dearth of direct evidence characterizing neural activity in response to VNS. A detailed understanding of the brain activity evoked by VNS across a range of stimulation parameters may guide selection of stimulation regimens for therapeutic use. In this study, we recorded neural activity in the LC and the mesencephalic trigeminal nucleus (Me5) in response to VNS over a broad range of current amplitudes, pulse frequencies, train durations, inter-train intervals, and pulse widths. Brief 0.5s trains of VNS drive rapid, phasic firing of LC neurons at 0.1mA. Higher current intensities and longer pulse widths drive greater increases in LC firing rate. Varying the pulse frequency substantially affects the timing, but not the total amount, of phasic LC activity. VNS drives pulse-locked neural activity in the Me5 at current levels above 1.2mA. These results provide insight into VNS-evoked phasic neural activity in multiple neural structures and may be useful in guiding the selection of VNS parameters to enhance clinical efficacy.

Effects of 3 Hz and 60 Hz Extremely Low Frequency Electromagnetic Fields on Anxiety-Like Behaviors, Memory Retention of Passive Avoidance and Electrophysiological Properties of Male Rats.

The effects of electromagnetic fields on biological organisms have been a controversial and also interesting debate over the past few decades, despite the wide range of investigations, many aspects of extremely low frequency electromagnetic fields (ELF/EMFs) effects including mechanism of their interaction with live organisms and also their possible biological applications still remain ambiguous. In the present study, we investigated whether the exposures of ELF/EMF with frequencies of 3 Hz and 60 Hz can affect the memory, anxiety like behaviors, electrophysiological properties and brain's proteome in rats. Male rats were exposed to 3 Hz and 60 Hz ELF/EMFs in a protocol consisting of 2 cycles of 2 h/day exposure for 4 days separated with a 2-day interval. Short term memory and anxiety like behaviors were assessed immediately, 1 and 2 weeks after the exposures. Effects of short term exposure were also assessed using electrophysiological approach immediately after 2 hours exposure. Behavioral test revealed that immediately after the end of exposures, locomotor activity of both 3 Hz and 60 Hz exposed groups significantly decreased compared to sham group. This exposure protocol had no effect on anxiety like behavior during the 2 weeks after the treatment and also on short term memory. A significant reduction in firing rate of locus coeruleus (LC) was found after 2 hours of both 3 Hz and 60 Hz exposures. Proteome analysis also revealed global changes in whole brain proteome after treatment. Here, some evidence regarding the fact that such exposures can alter locomotor activity and neurons firing rate in male rats were presented.

Long-term administration of the antidepressant vilazodone modulates rat brain monoaminergic systems

Vilazodone has high affinity for the human 5-hydroxytryptamine1A (h5-HT1A) receptor and for the serotonin transporter (5-HTT). A previous in vivo microdialysis experiment showed that a single administration of vilazodone, dose-dependently increases extracellular 5-HT but not norepinephrine (NE) or dopamine (DA) levels in rat medial prefrontal cortex and ventral hippocampus. The effects of vilazodone on monoaminergic systems were assessed using single-unit extracellular recordings and microiontophoresis in the rat brain. Following depletion of 5-HT with para-chlorophenylalanine methyl-ester hydrochloride (PCPA), vilazodone still suppressed neuronal firing of dorsal raphe nucleus (DRN) 5-HT neurons to a similar extent than controls, indicating that this inhibition is via 5-HT1A receptors activation. Following 2-day intraperitoneal administration of vilazodone (5 mg/kg/day), there was a significant decrease in 5-HT neuronal firing which recovered to baseline levels by day 14 of administration, likely due to 5-HT1A autoreceptor desensitization. Two- and 14-day administration of vilazodone decreased the mean firing and bursting activities of ventral tegmental area (VTA) DA neurons, while only its repeated administration significantly dampened the mean firing rate of locus coeruleus (LC) NE neurons. Vilazodone acted as an agonist at 5-HT1A receptors, while showing a 5-HTT blocking capacity when injected acutely. After repeated vilazodone regimen, while there was no change in sensitivity of 5-HT1A receptors, the enhancement in 5-HT transmission yielded an increase in the tonic activation of these receptors located in the hippocampus.

The effect of morphine on some electrophysiological parameters of paragigantocellularis and locus coeruleus nuclei interconnections

As one of the most important diffused brain modulatory systems, the nucleus locus coeruleus (LC) receives most of its afferents from the nucleus paragigantocellularis (PGi) and plays a major role in the control of drug dependence and some emotional and exciting states. For detailed investigation of the effect of morphine on relationship between these two brain stem nuclei, the activity of the rat (Sprague-Dawley) LC neurons was examined after being dependent on morphine in comparison with independent group. The activity of the nucleus LC neurons with and without the activity of nucleus PGi neurons was recorded using extracellular single unit recording technique. Lidocaine microinjection (1 ml, in 1-2 min, 2.5%) was used for inactivation of PGi. The PSTH (Peri-Stimulus Time Histogram) of the neuronal activity showed a decrease in neuronal firing rate of LC by 85.8±1.7% in morphine-dependent group (from 2.95 spike/sec to 0.64 spike/sec, n = 4) and by 48.3±4.3% (from 3.5 spike/sec to 1.94 spike/sec, n = 6) for independent one. The difference was significant (P

Locus coeruleus and dorsal raphe neuron activity and response to acute antidepressant administration in a rat model of Parkinson's disease

In addition to noradrenergic and serotonergic systems, dopaminergic neurotransmission seems to play an important role in the aetiopathogenesis of, and recovery from, depression. Moreover, the incidence of depression is higher in patients affected by diseases where the dopaminergic system is highly impaired, such us Parkinson's disease. Here, we investigated the effects of dopamine degeneration on the activity and response to antidepressants of locus coeruleus (LC) noradrenergic and dorsal raphe nucleus (DRN) serotonergic neurons. To this end, single-unit extracellular recordings were performed in control and 6-hydroxydopamine (6-OHDA)-lesioned animals. In this latter group, LC neurons showed a lower basal firing rate as well as less sensitivity to the administration of the serotonin reuptake inhibitor, fluoxetine. The rest of electrophysiological parameters and the response to the administration of the α2-adrenoceptor agonist, clonidine and the noradrenaline reuptake inhibitor, reboxetine remained unaltered. In the DRN, dopamine depletion did not modify the basal electrophysiological characteristics and the response to clonidine or fluoxetine administration. In contrast, the administration of reboxetine more efficiently induced an inhibitory effect in the lesioned group. In additional analyses it was observed that while in control animals, LC and DRN basal firing rate was significantly correlated, this relationship was lost after the 6-OHDA lesion. In conclusion, dopaminergic degeneration alters LC neuron basal activity, the relationship/synteny between both nuclei, and their response to antidepressants. These findings shed fresh light on our understanding of the role of dopamine in depression and the mechanism action of antidepressants.

Neonatal rearing conditions distinctly shape locus coeruleus neuronal activity, dendritic arborization, and sensitivity to corticotrophin-releasing factor

Early life events influence vulnerability to psychiatric illness. This has been modelled in rats and it has been demonstrated that different durations of maternal separation shape adult endocrine and behavioural stress reactivity. One system through which maternal separation may act is the locus coeruleus (LC)-norepinephrine system that regulates emotional arousal. Here we demonstrate that different durations of maternal separation have distinct effects on LC physiology and dendritic morphology. Rat pups were separated from the dam for 15 min/d (HMS-15) or 180 min/d (HMS-180) from post-natal days 2-14. Others were either undisturbed (HMS-0) or were vendor-purchased controls. LC characteristics were compared at age 22-35 d using whole-cell recordings in vitro. Cells were filled with biocytin for morphological analysis. LC neurons of HMS-180 rats were tonically activated compared to HMS-15 and control rats, with firing rates that were 2-fold higher than these groups. Corticotrophin-releasing factor (CRF) application did not further activate LC neurons of HMS-180 rats but increased LC firing rate in HMS-0 and control rats. LC neurons of HMS-15 rats were resistant to excitation by CRF. Maternal separation also affected LC dendritic morphology. LC dendrites of HMS-15 rats exhibited less branching and decreased total dendritic length, an effect that could decrease the probability of contacting limbic afferents that terminate in the pericoerulear region. This effect may provide a structural basis for an attenuated magnitude of emotional arousal. Together, these results demonstrate long-term consequences of early life events on the LC-norepinephrine system that may shape adult behaviour.

Effect of Vagus Nerve Stimulation on Serotonergic and Noradrenergic Transmission

Vagus nerve stimulation (VNS) is an antiepileptic treatment, which has recently shown promise as an antidepressant. Yet, its antidepressant mechanisms of action are unknown. Serotonergic [5-hydroxytryptamine (5-HT, serotonin)] and noradrenergic [norepinephrine (NE)] systems are involved in the pathophysiology of depression and in the mechanisms of action of antidepressants. The present study analyzes 5-HT and NE neuronal firing rates in their brainstem nuclei: the dorsal raphe nucleus (DRN) and locus coeruleus (LC), respectively. The basal firing rates in the DRN and LC were significantly increased after long-term treatments with VNS. After short-term VNS treatments, firing rates were significantly higher for LC (at 1 h and 3 days). As changes in their firing rate may have been due to altered autoreceptor sensitivities, the responses of autoreceptors to the acute administration of their respective agonists were assessed. However, no significant difference was seen in the DRN. No significant differences in dose response curves for 5-HT(1A) somatodendritic and alpha 2-adrenergic autoreceptors were noticed between long-term VNS and controls. VNS appears to have a novel mechanism of antidepressant action, enabling its effectiveness in treatment-resistant depression. LC firing rates significantly increase earlier than the DRN basal firing. As the LC has an excitatory influence on DRN, it is possible that the increased DRN firing rate is secondary to an initial increased LC firing rate from VNS.

Fluoxetine administration potentiates the effect of olanzapine on locus coeruleus neuronal activity

Background As many as 30% of individuals diagnosed with depression are nonresponsive to traditional antidepressant medication. Augmentation and combination strategies have emerged in an attempt to address this issue. Atypical antipsychotics (e.g., olanzapine), when added to a selective serotonin reuptake inhibitor (e.g., fluoxetine) have shown great promise in the treatment of these treatment-resistant patients. As of yet, the precise neural mechanisms responsible for the beneficial clinical effect of these combinations are not completely understood. Methods Separate groups of rats received either saline or fluoxetine (10 mg/kg/day) for 24 hours or 3 weeks via subcutaneously implanted osmotic pumps. The effects of either intravenous saline or olanzapine (.3, 1.0, or 3.0 mg/kg) on locus coeruleus (LC) neuronal activity were then assessed via extracellular single-unit recordings. Results Acute administration of olanzapine produced a significant elevation of the firing rate and burst firing of LC cells, and chronic, but not acute, administration of fluoxetine decreased baseline and burst firing of LC cells; however, when given in combination, an interaction of fluoxetine and olanzapine was observed, with olanzapine causing a significantly greater increase in LC firing rate and burst firing after acute and chronic administration of fluoxetine. Conclusions These results provide a potential neural mechanism for the beneficial clinical effects of the olanzapine/fluoxetine combination. The increase in baseline and burst firing of LC neurons in the groups receiving both fluoxetine and olanzapine would result in enhanced norepinephrine release in projection areas (e.g., prefrontal cortex), which could lead to a reduction in depressive symptoms.

Short-term effects of 3,4-methylenedioximethamphetamine on noradrenergic activity in locus coeruleus and hippocampus of the rat

This study was undertaken to investigate the effects of the administration of 3,4-methylenedioxymethamphetamine (MDMA) on the locus coeruleus firing rate, on the sensitivity of the α 2-adrenoceptors which regulate neuronal activity and on the in vivo tyrosine hydroxylase activity in hippocampus. The basal firing rate was not modified by either a single dose or repeated doses of MDMA, although the latter produced a shift to the right in the dose-response curve for clonidine-induced inhibition of the firing rate (ED 50 increased by 59%) and a reduction in tyrosine hydroxylase activity (20%) in the hippocampus. However, 8 days after the final dose α 2-adrenoceptor sensitivity and tyrosine hydroxylase activity had returned to control values. Our results show a desensitization of α 2-adrenoceptors in locus coeruleus and the existence of short-term changes in the noradrenergic system.

Functional and pharmacological characterization of the modulatory role of serotonin on the firing activity of locus coeruleus norepinephrine neurons

Previous studies, using in vivo extracellular unitary recordings in anaesthetized rats, have shown that the selective 5-HT 1A receptor antagonist WAY 100,635 suppressed the firing rate of locus coeruleus (LC) norepinephrine (NE) neurons and that this effect was abolished by lesioning 5-HT neurons. In the present experiments, the selective 5-HT 2A receptor antagonist MDL 100,907, while having no effect on the spontaneous firing activity of LC neurons in controls, was able to restore NE neuronal discharges following the injection of WAY 100,635. The 5-HT 1A receptor agonist 8-OH-DPAT enhanced the firing activity of NE neurons and this action was entirely dependent on intact 5-HT neurons, unlike the inhibitory effect of the 5-HT 2 receptor agonist DOI. Taken together, these data indicate that 5-HT 2A but not 5-HT 1A receptors controlling LC firing activity are postsynaptic to 5-HT neurons. Prolonged, but not subacute, administration of selective 5-HT reuptake inhibitors (SSRIs) produces a decrease in the spontaneous firing activity of LC NE neurons. MDL 100,907 partially reversed this suppressed firing activity of LC neurons in paroxetine-treated rats. Although the α 2-adrenoceptor antagonist idazoxan also enhanced the firing activity of NE neurons in paroxetine-treated rats, this increase was similar to that obtained in controls. In conclusion, prolonged SSRI treatment enhances a tonic inhibitory influence by 5-HT on LC neurons through postsynaptic 5-HT 2A receptors that are not located on NE neurons. A speculative neuronal circuitry accounting for these phenomena on LC NE activity is proposed.

Effects of corticotropin-releasing hormone on locus coeruleus neurons in vivo: a microdialysis study using a novel bilateral approach.

Stress-induced release of noradrenaline (NA) from locus coeruleus (LC) neurons is mainly regulated by corticotropin-releasing hormone (CRH). Tyrosine is a precursor of NA and plays an intriguing role in the regulation of NA release. We studied the effects of injecting CRH into the LC using a novel bilateral approach which relies on the mainly ipsilateral projections of LC neurons allowing stimulation of one hemisphere while using the other as control. To analyze the modification of the CRH effect, tyrosine was given intraperitoneally. A combination of CRH and its antagonist d-Phe was administered for validation of the specificity of CRH effects. Wistar rats were used in all experiments. Injections were made through fused silica capillaries implanted into both LCs and microdialysis samples were collected bilaterally from the prefrontal cortex (PFM) every 20 min for 1 h before and 3 h after injections. The effects of LC stimulation were investigated by determining 3-methoxy-4-hydroxyphenylglycol (MHPG) in the dialysates. Following CRH injection into one LC and contralateral infusion of artificial cerebrospinal fluid (aCSF), MHPG levels, which are indicative of NA release, increased only in the ipsilateral PFM. These effects were blocked by d-Phe. Simultaneous administration of tyrosine i.p. led to a significant prolongation of MHPG release. These data provide the first physiological evidence of unilateral LC projections with the bilateral stimulation design proving to be a very valuable tool for the study of LC firing rate, to decrease number of animals and time expenditure. Prolongation of MHPG release after tyrosine supplementation is most likely due to increased NA synthesis.

Potent excitatory influence of prefrontal cortex activity on noradrenergic locus coeruleus neurons

An influence of the prefrontal cortex on noradrenergic locus coeruleus neurons would have profound implications for the function of the locus coeruleus system. Although the medial prefrontal cortex does not substantially innervate the core of the nucleus locus coeruleus, evidence indicates that the medial prefrontal cortex projects to regions containing locus coeruleus dendrites; indirect medial prefrontal cortex-locus coeruleus projections are also possible. Here, we examined influences of prefrontal cortex activity on locus coeruleus firing rates by activating or inactivating the medial prefrontal cortex while recording impulse activity of locus coeruleus neurons extracellularly in anaesthetized rats. Most of our electrical stimulation experiments were conducted in rats which underwent lesions of the ascending dorsal bundle of noradrenergic fibres from the locus coeruleus to eliminate locus coeruleus projections to the prefrontal cortex, because antidromic activation of locus coeruleus from the prefrontal cortex affects even non-driven locus coeruleus neurons through collaterals. Single pulse stimulation (1 mA, 0.3-0.5 ms) of the dorsomedial (frontal region 2) or prelimbic region of the medial prefrontal cortex synaptically activated 13/16 (81%) or 16/56 (29%) locus coeruleus neurons, respectively. Train stimulation (20 Hz for 0.5 s) synaptically activated greater percentages of locus coeruleus cells, 11/12 cells (92%) for the dorsomedial prefrontal cortex, and 41150 cells (82%) for the prelimbic cortex. No inhibitory responses in the locus coeruleus were obtained with dorsomedial prefrontal stimulation, and weak inhibition was found in 16% of locus coeruleus cells with prelimbic stimulation. Electrical stimulation of more lateral frontal cortex (Frl area) had no effects on locus coeruleus activity. Chemical stimulation of the dorsomedial prefrontal cortex with l-glutamate (10 or 100 mM) or d, l-homocysteic acid (10 mM) phasically activated 15/26 (55%) locus coeruleus cells, and 15/68 cells (22%) with prelimbic stimulation; such activation was sometimes followed by long-lasting oscillatory activity. No locus coeruleus cells exhibited purely inhibitory responses with chemical stimulation of any prefrontal cortex site. Inactivation of the dorsomedial or prelimbic region of the prefrontal cortex with lidocaine microinjection (2%, 180 or 300 nl) reduced locus coeruleus firing rates in 6/10 (60%) or 7/19 (37%) locus coeruleus cells, respectively. In no case did lidocaine in any prefrontal cortex site activate a locus coeruleus neuron. These results indicate that the medial prefrontal cortex provides a potent excitatory influence on locus coeruleus neurons. The fact that inactivation of the medial prefrontal cortex suppressed locus coeruleus firing indicates that the medial prefrontal cortex also provides a resting tonic excitatory influence on locus coeruleus activity.

Agmatine does not have activity at α2-adrenoceptors which modulate the firing rate of locus coeruleus neurones: an electrophysiological study in rat

Agmatine (decarboxylated arginine) has been proposed as an endogenous ligand for non-adrenoceptor, imidazoline binding sites, but also binds to α 2-adrenoceptors. The interaction of agmatine with α 2-adrenoceptors was evaluated by studying the effect of agmatine on the firing rate of locus coeruleus (LC) neurones using extracellular recordings in anesthetized rats and rat brain slices. In vivo, local application of agmatine into the LC caused a slight and short-lasting increase in cell firing rate ( P < 0.005). In vitro, agmatine failed to change the firing rate of LC neurones nor did it antagonize the inhibitory effect of noradrenaline on these cells. Since α 2-adrenoceptors are known to inhibit the firing of LC cells, we conclude that agmatine does not have agonist or antagonist properties at α 2-adrenoceptors of these neurones.

Evidence that the acute behavioral and electrophysiological effects of bupropion (Wellbutrin) are mediated by a noradrenergic mechanism.

Bupropion (BW 323U66) has been considered a dopaminergic antidepressant based on its ability to inhibit the uptake of dopamine (DA) somewhat more selectively than it inhibits uptake of norepinephrine (NE) or serotonin (5-HT). This report describes new evidence that bupropion selectively inhibits firing rates of NE cells in the locus coeruleus (LC) at doses significantly lower than those that inhibit activity of midbrain DA cells or dorsal raphe 5-HT cells. The IC50 dose (13 mg/kg i.p.) for inhibition of LC firing produced plasma concentrations that were not significantly different from those generated by the ED50 in the Porsolt test (10 mg/kg i.p.). The fourfold higher dose needed to inhibit DA cell firing (IC50 = 42 mg/kg i.p.) was similar to the dose associated with locomotor stimulation in freely moving rats. Bupropion did not change the firing rates of 5-HT cells in the dorsal raphe nucleus at any dose. In both in vitro and in vivo tests, the metabolite 306U73 (hydroxybupropion), a weak inhibitor of NE uptake, was approximately equipotent to bupropion with regard to inhibition of LC cells. Another metabolite, 494U73, had no effect on LC firing rates over a wide range of doses. Because of species variation in metabolism, 306U73 was not detected in plasma of rats after i.v. doses of bupropion that inhibited LC firing. Only trace amounts of 306U73 were detected after bupropion dosing for the Porsolt test. Pretreatment with reserpine markedly depleted catecholamines and reduced (by 30-fold) the potency of bupropion to inhibit LC firing.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordinate regulation of the cyclic AMP system with firing rate and expression of tyrosine hydroxylase in the rat locus coeruleus: effects of chronic stress and drug treatments.

Recent studies have demonstrated that chronic stress increases the firing rate and expression of tyrosine hydroxylase (TH) in neurons of the locus coeruleus (LC), the major noradrenergic nucleus in brain. The present study was undertaken to examine the influence of chronic stress and other treatments known to influence the activity of LC neurons on the cyclic AMP (cAMP) second messenger system in these neurons. Chronic (5 days) cold exposure significantly increased levels of TH immunoreactivity in the LC, as previously reported, but not in substantia nigra (SN) or ventral tegmentum (VT), two dopaminergic nuclei studied for comparison. Chronic cold exposure increased levels of cAMP-dependent protein kinase activity in soluble, but not particulate, fractions of the LC, and increased basal and GTP- and forskolin-stimulated adenylate cyclase activity in this brain region. In contrast, levels of the protein kinase and adenylate cyclase in VT, SN, and frontal cortex were not significantly influenced by cold exposure. To study further the relationship between regulation of LC firing rate, TH expression, and the cAMP system in the LC, other treatments known to influence TH were examined. Reserpine treatment, shown previously to increase levels of TH, was found to increase both LC firing rate and levels of soluble cAMP-dependent protein kinase activity in the LC. 6-Hydroxydopamine, shown previously to increase levels of TH and firing rate of LC neurons, also increased soluble levels of protein kinase activity. Other treatments known to either increase (adrenalectomy) or decrease (chronic imipramine) levels of TH in the LC were also found to increase or decrease, respectively, levels of cAMP-dependent protein kinase activity in this brain region.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of locus coeruleus (LC) neurones by cholera toxin: Mediation by cAMP-dependent protein kinase

There is evidence that the tonic pacemaker activity of the noradrenergic pacemaker neurones of the locus coeruleus (LC) depends on endogenous cAMP acting via protein kinase A and its phosphorylation pathway. In this study, we tested the effect of cholera toxin, which produces persistent activation of G s, on LC firing rates. Bath applied cholera toxin (holotoxin) increased LC firing rates after a lag of 50–110 min. Intracellularly applied A-subunit ( active-subunit) but not the B-subunit ( binding-subunit) of cholera toxin via low resistance patch electrodes mimicked the excitatory actions of bath-applied holotoxin but without its lag period. The effects of both bath-applied and intracellularly applied cholera toxin A-subunit were blocked by intracellular applications of a specific cAMP-dependent protein kinase inhibitor (PKI 5–24). We conclude that persistent activation of G s by cholera toxin (A-subunit) increases LC firing rates via the cAMP-dependent protein phosphorylation pathway.