Firing Rate Of Locus Coeruleus Neurons Research Articles

Inhibition of rat locus coeruleus neurons by prostaglandin E2 EP3 receptors: pharmacological characterization ex vivo.

Prostaglandin E2 (PGE2) is an inflammatory mediator synthesized by the brain constitutive cyclooxygenase enzyme. PGE2 binds to G protein-coupled EP1-4 receptors (EP1 to Gq, EP2,4 to Gs, and EP3 to Gi/o). EP2, EP3 and EP4 receptors are expressed in the locus coeruleus (LC), the main noradrenergic nucleus in the brain. EP3 receptors have been explored in the central nervous system, although its role regulating the locus coeruleus neuron activity has not been pharmacologically defined. Our aim was to characterize the function of EP3 receptors in neurons of the LC. Thus, we studied the effect of EP3 receptor agonists on the firing activity of LC cells in rat brain slices by single-unit extracellular electrophysiological techniques. The EP3 receptor agonist sulprostone (0.15nM-1.28µM), PGE2 (0.31nM-10.2µM) and the PGE1 analogue misoprostol (0.31nM-2.56µM) inhibited the firing rate of LC neurons in a concentration-dependent manner (EC50 = 15nM, 110nM, and 51nM, respectively). The EP3 receptor antagonist L-798,106 (3-10µM), but not the EP2 (PF-04418948, 3-10µM) or EP4 (L-161,982, 3-10µM) receptor antagonists, caused rightward shifts in the concentration-effect curves for the EP3 receptor agonists. Sulprostone-induced effect was attenuated by the Gi/o protein blocker pertussis toxin (pertussis toxin, 500ngml-1) and the inhibitors of inwardly rectifying potassium channels (GIRK) BaCl2 (300µM) and SCH-23390 (15µM). In conclusion, LC neuron firing activity is regulated by EP3 receptors, presumably by an inhibitory Gi/o protein- and GIRK-mediated mechanism.

Acute morphine injection persistently affects the electrophysiological characteristics of rat locus coeruleus neurons

Administration of morphine is associated with critical complications in clinic which primarily includes the development of dependence and tolerance even following a single dose (acute) exposure. Behavioral and electrophysiological studies support the significant role of locus coeruleus (LC) neurons in tolerance and dependence following chronic morphine exposure. The current study was designed to explore the electrophysiological properties of the LC neurons following acute morphine exposure. In-vitro whole-cell patch-clamp recordings were performed in LC neurons 24 h after intraperitoneal morphine injection. Acute morphine injection significantly decreased the spontaneous firing rate of LC neurons, the rising and decay slopes of action potentials, and consequently increased the action potential duration. In addition, morphine treatment did not alter the rheobase current and first spike latency while affected the inhibitory postsynaptic currents elicited in response to orexin-A. In fact, single morphine exposure could inhibit the disinhibitory effect of orexin-A on LC neurons.

Synergistic effect of orexin-glutamate co-administration on spontaneous discharge rate of locus coeruleus neurons in morphine-dependent rats

Various intrinsic and extrinsic factors can increase the spontaneous discharge rate of locus coeruleus (LC) neurons. Among the extrinsic ones, orexinergic neurons of the lateral hypothalamus (LH) send widespread projections to LC region. Accumulating evidence support the involvement of glutamate in mediating the excitatory effect of orexin-A on LC neurons. In addition, both orexinergic and glutamatergic systems have been shown to play critical roles in molecular changes underlying the development of morphine dependence. The present study was designed to investigate the interaction between orexin-A and glutamate in modulating the firing rate of LC neurons. Regarding the role of both orexinergic and glutamatergic systems in morphine dependence, this effect was also investigated in morphine dependent rats. For this purpose, spontaneous discharge rate of LC neurons was recorded using the whole-cell patch clamp recording method in presence of orexin-A, glutamate or orexin-A plus glutamate in acutely prepared brain slices. Results indicated that superfusion of either orexin-A or glutamate enhances the firing rate of LC neurons in both dependent and non-dependent rats. However, co-application of orexin-A and glutamate elicited a significant synergism in enhancement of firing rate only in morphine dependent animals. In conclusion, it seems that development of morphine dependence promotes adaptations in locus coeruleus neurons that potentiate the orexin-glutamate interaction.

Central antagonism of orexin type-1 receptors attenuates the development of morphine dependence in rat locus coeruleus neurons

Prolonged use/abuse of opioid agonists leads to development of severe dependence to these drugs. Orexin-A has a crucial role in development of morphine dependence. The locus coeruleus (LC) is implicated in the expression of morphine withdrawal signs. Hyperactivity of LC neurons as well as increased intracellular cyclic adenosine monophosphate (cAMP) level temporally corresponds to the expression of opioid withdrawal behaviors. In this study the effect of central OX1R blockade on neuronal activity and cAMP content of LC neurons was investigated following naloxone administration in morphine-dependent rats. Male Wistar rats weighing 250–300g were used in this study. To induce morphine dependence, morphine was injected intraperitoneally (10mg/kg, i.p.) once a day for seven consecutive days. A selective OX1R antagonist (SB-334867) was microinjected into the cerebral ventricle (10µg/10µl) immediately before each morphine injection. The activity of LC neurons was investigated using in vivo extracellular single-unit recording on day 8 and naloxone (2mg/kg, i.p.) was administered after 10-min baseline recording. In addition, immunohistofluorescence method was used to measure the effect of naloxone on coerulear cAMP level. Chronic morphine injection induced morphine dependence in LC neurons which was revealed as a significant increase in LC neuronal firing rate in response to naloxone. The results of this study indicated that SB-334867 administration prior to each morphine injection prevents naloxone-elicited neuronal activation within the LC. In addition, naloxone injection enhanced the cAMP concentration in LC neurons of morphine-dependent animals and this effect was significantly reduced by OX1R blockade.

Locus coeruleus neuronal activity correlates with behavioral response to acute and chronic doses of methylphenidate (Ritalin) in adolescent rats.

The objective of this study is to gain insight into the behavioral and neuronal changes induced by acute and chronic methylphenidate (MPD) administration. Specifically, there is limited knowledge of the effects of MPD on the locus coeruleus (LC), the main site of norepinephrine synthesis in the brain. In this study, LC neuronal firing rate was recorded simultaneously with locomotor activity in freely moving adolescent rats. Adolescent rats were chosen to mimic the age group in humans most affected by MPD exposure. Following acute dose of 0.6, 2.5 or 10mg/kg MPD, all rats showed an increase in locomotor activity. However, in response to chronic MPD doses, individual rats showed either a further increase or decrease in their locomotor activity as compared to the effect initiated by the acute dose-expressing either behavioral sensitization or tolerance, respectively. The LC neuronal recordings from animals expressing behavioral sensitization showed that the majority of units responded to chronic MPD exposure by further increasing firing rates as compared to the initial response to the acute MPD exposure. For the LC neuronal units recorded from animals expressing behavioral tolerance, however, the majority of the units responded to chronic exposure by attenuating or no significant effect on their firing rate as compared to the acute MPD exposure. This observation indicates a correlation between the LC neuronal responses and behavioral activity to chronic MPD exposure. The study shows that LC participates in the effect of MPD and the behavioral expression of sensitization and tolerance to chronic exposure of MPD.

Corticotropin releasing factor dose-dependently modulates excitatory synaptic transmission in the noradrenergic nucleus locus coeruleus.

The noradrenergic nucleus locus coeruleus (LC) is critically involved in the stress response and receives afferent input from a number of corticotropin releasing factor (CRF) containing structures. Several invivo and invitro studies in rat have shown that CRF robustly increases the firing rate of LC neurons in a dose-dependent manner. While it is known that these increases are dependent on CRF receptor subtype 1 and mediated by effects of cAMP intracellular signaling cascades on potassium conductance, the impact of CRF on synaptic transmission within LC has not been clarified. In the present study, we used whole-cell patch clamp electrophysiology to assess how varying concentrations of bath-applied CRF affect AMPA-receptor dependent spontaneous excitatory post-synaptic currents (sEPSCs). Compared to vehicle, 10, 25, and 100nm CRF had no significant effects on any sEPSC parameters. Fifty nanomolar CRF, however, significantly increased sEPSC amplitude, half-width, and charge transfer, while these measures were significantly decreased by 200nm CRF. These observations suggest that stress may differentially affect ongoing excitatory synaptic transmission in LC depending on how much CRF is released from presynaptic terminals. Combined with the well-documented effects of CRF on membrane properties and spontaneous LC discharge, these observations may help explain how stress and CRF release are able to modulate the signal to noise ratio of LC neurons. These findings have implications for how stress affects the fidelity of signal transmission and information flow through LC and how it might impact norepinephrine release in the CNS.

An optogenetic mouse model of rett syndrome targeting on catecholaminergic neurons.

Rett syndrome (RTT) is a neurodevelopmental disorder affecting multiple functions, including the norepinephrine (NE) system. In the CNS, NE is produced mostly by neurons in the locus coeruleus (LC), where defects in intrinsic neuronal properties, NE biosynthetic enzymes, neuronal CO2 sensitivity, and synaptic currents have been reported in mouse models of RTT. LC neurons in methyl-CpG-binding protein 2 gene (Mecp2) null mice show a high rate of spontaneous firing, although whether such hyperexcitability might increase or decrease the NE release from synapses is unknown. To activate the NEergic axonal terminals selectively, we generated an optogenetic mouse model of RTT in which NEergic neuronal excitability can be manipulated with light. Using commercially available mouse breeders, we produced a new strain of double-transgenic mice with Mecp2 knockout and channelrhodopsin (ChR) knockin in catecholaminergic neurons. Several RTT-like phenotypes were found in the tyrosine hydroxylase (TH)-ChR-Mecp2(-/Y) mice, including hypoactivity, low body weight, hindlimb clasping, and breathing disorders. In brain slices, optostimulation produced depolarization and an increase in the firing rate of LC neurons from TH-ChR control mice. In TH-ChR control mice, optostimulation of presynaptic NEergic neurons augmented the firing rate of hypoglossal neurons (HNs), which was blocked by the α-adrenoceptor antagonist phentolamine. Such optostimulation of NEergic terminals had almost no effect on HNs from two or three TH-ChR-Mecp2(-/Y) mice, indicating that excessive excitation of presynaptic neurons does not benefit NEergic modulation in mice with Mecp2 disruption. These results also demonstrate the feasibility of generating double-transgenic mice for studies of RTT with commercially available mice, which are inexpensive, labor/time efficient, and promising for cell-specific stimulation. © 2016 Wiley Periodicals, Inc.

The ventrolateral preoptic nucleus is required for propofol-induced inhibition of locus coeruleus neuronal activity.

The mechanisms underlying the unconsciousness of general anesthesia are not completely understood. Accumulating evidence indicates the ventrolateral preoptic nucleus (VLPO) in the endogenous sleep circuits may contribute to loss of consciousness (LOC) induced by GABA-enhancing anesthetics. However, there are few studies that look into distinct sleep pathway in the sleep-wake system. In the neural pathway from VLPO to the locus coeruleus (LC), we compared the inhibition effect of propofol on the LC activity before and after VLPO lesion in vivo rats. Systemic administration of propofol (20 mg/kg, i.p.) in normal rats caused a fast and obvious inhibition of LC neurons spontaneous firing (from 0.24 ± 0.06 to 0.12 ± 0.03 Hz). The LC neuronal firing rate of VLPO lesion rats only decreased to 0.18 ± 0.05 Hz (P = 0.021 vs. non-VLPO rats) after the propofol injection, and the time to reach the maximal inhibition level was also prolonged in VLPO lesion rats (2.3 ± 0.7 vs. 5.8 ± 1.2 min, P = 0.037). Microinjections of a selective GABAA receptor antagonist (SR95531) into the LC fully reversed the inhibitory effect of propofol on the LC neuronal activity, but did not significantly affect the latency to loss of righting reflex of rats after propofol administration (3.4 ± 0.9 vs. 3.7 ± 1.2 min, P = 0.639). Our results indicated that VLPO is necessary for the propofol-induced inhibition of LC activity, but the LC may not play an important role in the propofol-induced LOC.

Chemosensitive locus coeruleus neurons in the Savannah monitor lizard, Varanus exanthematicus (879.13)

Savannah monitor lizards, Varanus exanthematicus, are unusual among air‐breathing, ectothermic vertebrates by maintaining arterial pH nearly constant during changes in body temperature (‐0.003 U °C‐1) in contrast to the typical α‐stat regulating strategy (‐0.015 to ‐0.017 U °C‐1) of most other ectotherms. Given the importance of pH in the control of ventilation, we examined the CO2/H+ sensitivity of neurons from the locus coeruleus (LC) region of Savannah monitor lizard brainstems. Whole‐cell patch clamp electrophysiology was used to record membrane voltage (Vm) in neurons within the LC region in brainstem slices; changes in firing rate of LC neurons were calculated from action potential recordings. Upon exposure to hypercapnic acidosis (HA) at 20°C, 60% of neurons significantly decreased firing rate (1.8±0.3 vs. 0.6±0.1 Hz; P<0.05). The percentage of LC neurons inhibited by HA is high at 20°C compared with published percentages from bullfrogs and rats. Increasing temperature to 30°C abolished the inhibited response to HA (2.4±0.9 Hz vs. 2.6±1.0 Hz; P=1.0). The large proportion of neurons inhibited by HA at 20°C may provide a mechanism whereby retention of whole body CO2 stores with cooling enables the pH‐stat regulation strategy of Varanus exanthematicus.Grant Funding Source: Supported by NSF IOS 1257338

Temperature influences neuronal activity and CO2/pH sensitivity of locus coeruleus neurons in the bullfrog,Lithobates catesbeianus

The locus coeruleus (LC) is a chemoreceptive brain stem region in anuran amphibians and contains neurons sensitive to physiological changes in CO2/pH. The ventilatory and central sensitivity to CO2/pH is proportional to the temperature in amphibians, i.e., sensitivity increases with increasing temperature. We hypothesized that LC neurons from bullfrogs, Lithobates catesbeianus, would increase CO2/pH sensitivity with increasing temperature and decrease CO2/pH sensitivity with decreasing temperature. Further, we hypothesized that cooling would decrease, while warming would increase, normocapnic firing rates of LC neurons. To test these hypotheses, we used whole cell patch-clamp electrophysiology to measure firing rate, membrane potential (V(m)), and input resistance (R(in)) in LC neurons in brain stem slices from adult bullfrogs over a physiological range of temperatures during normocapnia and hypercapnia. We found that cooling reduced chemosensitive responses of LC neurons as temperature decreased until elimination of CO2/pH sensitivity at 10°C. Chemosensitive responses increased at elevated temperatures. Surprisingly, chemosensitive LC neurons increased normocapnic firing rate and underwent membrane depolarization when cooled and decreased normocapnic firing rate and underwent membrane hyperpolarization when warmed. These responses to temperature were not observed in nonchemosensitive LC neurons or neurons in a brain stem slice 500 μm rostral to the LC. Our results indicate that modulation of cellular chemosensitivity within the LC during temperature changes may influence temperature-dependent respiratory drive during acid-base disturbances in amphibians. Additionally, cold-activated/warm-inhibited LC neurons introduce paradoxical temperature sensitivity in respiratory control neurons of amphibians.

Functional and morphological characterization of glutamate transporters in the rat locus coeruleus

Excitatory amino acid transporters (EAATs) in the CNS contribute to the clearance of glutamate released during neurotransmission. The aim of this study was to explore the role of EAATs in the regulation of locus coeruleus (LC) neurons by glutamate. We measured the effect of different EAAT subtype inhibitors/enhancers on glutamate- and KCl-induced activation of LC neurons in rat slices. EAAT2-3 expression in the LC was also characterized by immunohistochemistry. The EAAT2-5 inhibitor DL-threo-β-benzyloxaspartic acid (100 μM), but not the EAAT2, 4, 5 inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (100 μM) or the EAAT2 inhibitor dihydrokainic acid (DHK; 100 μM), enhanced the glutamate- and KCl-induced activation of the firing rate of LC neurons. These effects were blocked by ionotropic, but not metabotrobic, glutamate receptor antagonists. DHK (100 μM) was the only EAAT inhibitor that increased the spontaneous firing rate of LC cells, an effect that was due to inhibition of EAAT2 and subsequent AMPA receptor activation. Chronic treatment with ceftriaxone (200 mg·kg(-1) i.p., once daily, 7 days), an EAAT2 expression enhancer, increased the actions of glutamate and DHK, suggesting a functional impact of EAAT2 up-regulation on the glutamatergic system. Immuhistochemical data revealed the presence of EAAT2 and EAAT3 surrounding noradrenergic neurons and EAAT2 on glial cells in the LC. These results remark the importance of EAAT2 and EAAT3 in the regulation of rat LC by glutamate. Neuronal EAAT3 would be responsible for terminating the action of synaptically released glutamate, whereas glial EAAT2 would regulate tonic glutamate concentrations in this nucleus.

Synaptic interactions between perifornical lateral hypothalamic area, locus coeruleus nucleus and the oral pontine reticular nucleus are implicated in the stage succession during sleep-wakefulness cycle

The perifornical area in the posterior lateral hypothalamus (PeFLH) has been implicated in several physiological functions including the sleep-wakefulness regulation. The PeFLH area contains several cell types including those expressing orexins (Orx; also known as hypocretins), mainly located in the PeF nucleus. The aim of the present study was to elucidate the synaptic interactions between Orx neurons located in the PeFLH area and different brainstem neurons involved in the generation of wakefulness and sleep stages such as the locus coeruleus (LC) nucleus (contributing to wakefulness) and the oral pontine reticular nucleus (PnO) nucleus (contributing to REM sleep). Anatomical data demonstrated the existence of a neuronal network involving the PeFLH area, LC, and the PnO nuclei that would control the sleep-wake cycle. Electrophysiological experiments indicated that PeFLH area had an excitatory effect on LC neurons. PeFLH stimulation increased the firing rate of LC neurons and induced an activation of the EEG. The excitatory effect evoked by PeFLH stimulation in LC neurons was blocked by the injection of the Orx-1 receptor antagonist SB-334867 into the LC. Similar electrical stimulation of the PeFLH area evoked an inhibition of PnO neurons by activation of GABAergic receptors because the effect was blocked by bicuculline application into the PnO. Our data also revealed that the LC and PnO nuclei exerted a feedback control on neuronal activity of PeFLH area. Electrical stimulation of LC facilitated firing activity of PeFLH neurons by activation of catecholaminergic receptors whereas PnO stimulation inhibited PeFLH neurons by activation of GABAergic receptors. In conclusion, Orx neurons of the PeFLH area seem to be an important organizer of the wakefulness and sleep stages in order to maintain a normal succession of stages during the sleep-wakefulness cycle.

Regulation of central noradrenergic activity by 5-HT3 receptors located in the locus coeruleus of the rat

A functional interaction between serotonergic and noradrenergic systems has been shown in the locus coeruleus (LC). Noradrenaline (NA) levels in the prefrontal cortex (PFC) are dependent on the firing rate of LC neurons, which is controlled by α2 adrenoceptors (α2ADR). The aim of the present study was to investigate the role of 5-HT3 receptors (5HT3R) in the modulation of central noradrenergic activity. We measured extracellular NA concentrations in the LC and PFC by dual-probe microdialysis in awake rats and the firing rate of LC neurons by electrophysiological techniques in vitro. Administration of the 5HT3R agonists SR57227 (1–100 μM) and m-chlorophenylbiguanide (mCPBG, 1–100 μM) into the LC increased NA in this nucleus (Emax = 675 ± 121% and Emax = 5575 ± 1371%, respectively) and decreased NA in the PFC (Emax = −49 ± 6% and Emax = −25 ± 11%, respectively). Administration of the 5HT3R antagonist Y25130 (50 μM) into LC attenuated SR57227 effect in the LC (Emax = 323 ± 28%) and PFC (Emax = −37 ± 7%). The α2ADR antagonist RS79948 (1 μM) blocked the SR57227 effect in the PFC but it did not change the effect in the LC (Emax = 677 ± 202%). In electrophysiological assays, both mCPBG (1–10 μM) and SR57227 (1–10 μM) reduced the firing rate of about 50% of tested LC neurons (maximal effect = −37 ± 2% and −31 ± 4%, respectively); this effect was partially blocked by Y25130 (50 μM). Administration of RS79948 (1 μM) reversed the inhibition induced by mCPBG. Competition radioligand assays against [3H]UK14304 and [3H]RX821002 (α2ADR selective drugs) in the rat brain cortex showed a very weak affinity of SR57227 for α2ADR, whereas the affinity of mCPBG for α2ADR was 17-fold higher than that of SR57227 for α2ADR. The present results suggest that 5HT3R stimulate NA release in the LC, which promotes simultaneously a decrease in the firing rate of LC neurons through α2ADR and then a decrease of NA release in terminal areas such as the PFC.

Ovarian‐Steroid Modulation of Locus Coeruleus Activity in Female Rats: Involvement in Luteinising Hormone Regulation

The noradrenergic nucleus locus coeruleus (LC) has been reported to regulate luteinising hormone (LH) secretion in female rats. Both oestrogen and progestin receptors have been demonstrated in LC neurones, suggesting that these cells are possibly responsive to variations in circulating levels of ovarian steroids. We therefore evaluated changes in the activity of LC neurones during the oestrous cycle and after ovarian-steroid treatment in ovariectomised (OVX) rats, as determined by immunoreactivity to Fos-related antigens (FRA), which comprises all of the known members of the Fos family. Effects of ovarian steroids on the firing rate of LC neurones were also determined in a slice preparation. The number of FRA/tyrosine hydroxylase (TH)-immunoreactive (ir) neurones in the LC increased from 14.00-16.00 h on pro-oestrus, coinciding with the onset of the LH surge and rise in plasma progesterone. FRA immunoreactivity was unaltered during dioestrus. Oestradiol-treated OVX rats (OVX+E) displayed marked reduction in FRA/TH-ir neurones in LC compared to oil-treated OVX rats. Accordingly, oestradiol superfusion significantly reduced the spontaneous firing rate of LC neurones in slices from OVX rats. Compared to OVX+E, oestradiol-treated rats injected with progesterone at 08.00 h (OVX+EP) exhibited higher number of FRA/TH-ir neurones in the LC at 10.00 h and 16.00 h, and great amplification of the LH surge. Bath application of progesterone significantly increased the spontaneous firing rate of OVX+E LC neurones. Our data suggest that ovarian steroids may physiologically modulate the activity of LC neurones in females, with possible implications for LH secretion. Moreover, oestradiol and progesterone appear to exert opposite and complementary effects (i.e. whereas oestradiol inhibits, progesterone, after oestradiol priming, stimulates LC activity).

Chronic cold exposure increases RGS7 expression and decreases α2‐autoreceptor‐mediated inhibition of noradrenergic locus coeruleus neurons

Chronic stress exposure alters the central noradrenergic neurons originating from the locus coeruleus (LC). Previously, we demonstrated that evoked increases in the firing rate of LC neurons and their release of norepinephrine are enhanced following chronic cold exposure. In the present studies, we tested the hypothesis that reduced feedback inhibition of LC neurons might underlie these alterations in LC activity by examining the effect of alpha(2)-autoreceptor stimulation on LC activity in chronically stressed rats using in vivo and in vitro single unit recordings. Given that regulators of G-protein signaling (RGS) proteins can impact the coupling of alpha(2)-autoreceptors to downstream signaling cascades, we also explored the expression of several RGS proteins following chronic stress exposure. We observed that the alpha(2)-autoreceptor-evoked inhibition of LC neurons was reduced and that the expression of RGS7 was increased following chronic stress exposure. Finally, we demonstrated that intracellular administration of RGS7 via patch clamp electrodes mimicked the stress-induced decrease in clonidine-evoked autoreceptor-mediated inhibition. These novel data provide a mechanism to explain how chronic stress-induced alterations in receptor coupling can result in changes in alpha(2)-autoreceptor control of noradrenergic function throughout the central nervous system, potentially leading to alterations in anxiety-related behaviors, and may suggest novel therapeutic targets for the treatment of mood and anxiety disorders.

Excitatory regulation of noradrenergic neurons by l-arginine/nitric oxide pathway in the rat locus coeruleus in vivo

To elucidate conflicting findings about the role of L-arginine/nitric oxide (NO) pathway in the locus coeruleus (LC), we investigated the effects of different drugs affecting NO concentrations by single-unit extracellular recordings from LC neurons in vivo and in vitro. In anesthetized rats, central (3.8-15.3 nmol i.c.v.) and local (16.5-66 pmol into the LC) administrations of the NO donor sodium nitroprusside, but not those of the inactive analogue potassium ferricyanide (16.5-66 pmol into the LC), increased by 65-84% the firing rate of LC neurons. In brain slices, low concentrations (50-200 microM) of diethylamine/NO complex, a short-lived NO releaser, also increased the neuron firing rate, although higher drug concentrations (400-800 microM) caused slowly reversible reductions of the firing activity. On the other hand, the NO synthase inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) (148-371 nmol i.c.v.) and N(omega)-nitro-L-arginine (L-NA) (46 nmol i.c.v.) gradually decreased the firing rate of LC neurons, whereas the NO synthase substrate L-arginine (0.71-1.42 micromol i.c.v. and 0.6-4.8 nmol into the LC) increased the neuron activity. The latter effect was not mimicked by the vehicle or the less active isomer D-arginine (0.6-4.8 nmol into the LC). Unexpectedly, pretreatment with high concentrations of L-NAME (371 nmol and 18.5 micromol i.c.v.) or L-NA (45.6 nmol i.c.v. and 0.24 nmol into the LC) failed to block the effect of L-arginine. The glutamate receptor antagonist kynurenic acid (1 micromol i.c.v.) strongly reduced the effect of L-arginine but not that of sodium nitroprusside. These data confirm in vivo a direct excitatory effect of NO on LC neurons and suggest a tonic regulation of noradrenergic neurons by NO in vivo. L-arginine also excites LC neurons, but this effect may be caused by a nitric-oxide-unrelated glutamate-receptor-mediated mechanism.

Effect of intracellular pH clamping on the response to hypercapnia of locus coeruleus (LC) neurons

Central chemosensitive neurons increase their firing rate (FR) upon exposure to hypercapnia. Currently, pHi is believed to be the major signal in the chemosensitive (CS) response; however, a new model has been proposed suggesting that multiple factors (such as Ca++, CO2, pHi, and pHo) contribute to the CS response. While there is evidence for a significant role of pHi in the CS response, we hypothesize that hypercapnic acidosis (HA) can still increase FR even with no change in pHi. To test this hypothesis, we developed a method to clamp pHi using rapid diffusion of the weak acid, acetic acid, through the cell membrane according to the relationship: pHi = pHo + log ([C2H3O2] i/[C2H3O2] o). Pontine brainstem slices (300 μm) were cut from neonatal rat pups (P4-P16). FR of LC neurons was measured with whole cell patch pipettes, pHi was measured by fluorescence imaging using the dye pyranine, and neurons were loaded from the patch pipette with 50 mM acetate. Extracellular acetate was varied to set pHi at desired values using the weak acid diffusion technique. We eliminated pHi decreases (0.07 ± 0.06 pH unit, p>0.05, n=8) of LC neurons upon exposure to HA (15% CO2, pHo 7.0) compared to the normal response (0.25 ± 0.03 pH unit, p<0.05, n=20). Despite pHi clamping, HA induced an increase in FR (CS index = 144 ± 14%, n=7) similar to controls (140 ± 5%, n=17). While a decrease in pHi is sufficient to elicit the chemosensitive response, these data indicate that intracellular acidification is not required. A chemosensitive response to hypercapnia without a change in pHi strongly supports the multiple factors model of chemosensitivity. [NIH grants RO1 HL56683 and F32 HL80877].

Methyl parathion increases neuronal activities in the rat locus coeruleus

Exposure to organophosphate insecticides induces undesirable behavioral changes in humans, including anxiety and irritability, depression, cognitive disturbances and sleep disorders. Little information currently exists concerning the neural mechanisms underlying such behavioral changes. The brain stem locus coeruleus (LC) could be a mediator of organophosphate insecticide-induced behavioral toxicities since it contains high levels of acetylcholinesterase and is involved in the regulation of the sleep-wake cycle, attention, arousal, memory, and pathological processes, including anxiety and depression. In the present study, using a multi-wire recording technique, we examined the effects of methyl parathion, a commonly used organophosphate insecticide, on the firing patterns of LC neurons in rats. Systemic administration of a single dose of methyl parathion (1 mg/kg, i.v.) increased the spontaneous firing rates of LC neurons by 240% but did not change the temporal relationships among the activities of multiple LC neurons. This dose of methyl parathion induced a 50% decrease in blood acetylcholinesterase activity and a 48% decrease in LC acetylcholinesterase activity. The methyl parathion-induced excitation of LC neurons was reversed by administration of atropine sulfate, a muscarinic receptor antagonist, indicating an involvement of muscarinic receptors. The methyl parathion-induced increase in LC neuronal activity returned to normal within 30 min while the blood acetylcholinesterase activity remained inhibited for over 1 h. These data indicate that methyl parathion treatment can elicit excitation of LC neurons. Such excitation could contribute to the neuronal basis of organophosphate insecticide-induced behavioral changes in human.

Role of intracellular and extracellular pH in the chemosensitive response of rat locus coeruleus neurones.

The chemosensitive response of locus coeruleus (LC) neurones to changes in intracellular pH (pH(i)), extracellular pH (pH(o)) and molecular CO(2) were investigated using neonatal rat brainstem slices. A new technique was developed that involves the use of perforated patch recordings in combination with fluorescence imaging microscopy to simultaneously measure pH(i) and membrane potential (V(m)). Hypercapnic acidosis (15 % CO(2), pH(o) 6.8) resulted in a maintained fall in pH(i) of 0.31 pH units and a 93 % increase in the firing rate of LC neurones. On the other hand, isohydric hypercapnia (15 % CO(2), 77 mM HCO(3)(-), pH(o) 7.45) resulted in a smaller and transient fall in pH(i) of about 0.17 pH units and an increase in firing rate of 76 %. Acidified Hepes (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid)-buffered medium (pH(o) 6.8) resulted in a progressive fall in pH(i) of over 0.43 pH units and an increase in firing rate of 126 %. Isosmotic addition of 50 mM propionate to the standard HCO(3)(-)-buffered medium (5 % CO(2), 26 mM HCO(3)(-), pH(o) 7.45) resulted in a transient fall in pH(i) of 0.18 pH units but little increase in firing rate. Isocapnic acidosis (5 % CO(2), 7 mM HCO(3)(-), pH(o) 6.8) resulted in a slow intracellular acidification to a maximum fall of about 0.26 pH units and a 72 % increase in firing rate. For all treatments, the changes in pH(i) preceded or occurred simultaneously with the changes in firing rate and were considerably slower than the changes in pH(o). In conclusion, an increased firing rate of LC neurones in response to acid challenges was best correlated with the magnitude and the rate of fall in pH(i), indicating that a decrease in pH(i) is a major part of the intracellular signalling pathway that transduces an acid challenge into an increased firing rate in LC neurones.

Morphine Induces Synchronous Oscillatory Discharges in the Rat Locus Coeruleus

The noradrenergic locus coeruleus (LC) plays a role in opioid dependence and withdrawal. In the present study, using a multiple-electrode recording technique that allowed several LC neurons to be recorded simultaneously over long time periods, LC neuronal activities were recorded before and after intracerebroventricular injection of morphine (26 nmol) under halothane anesthesia. We found that morphine did not simply decrease firing rates of LC neurons, as reported in earlier studies, but that it induced persistent oscillatory discharges in 49% (87 of 178) of the LC neurons recorded. Cross-correlation analysis revealed that almost all LC neurons (86 of 87) that exhibited oscillatory discharges were synchronized with at least one other neuron. When stated in terms of simultaneously recorded neuron pairs, 59% (292 of 492) of the oscillatory neuron pairs discharged synchronously. The morphine-induced synchronous oscillation began at approximately 10 min after morphine injection, reached its peak in approximately 20-30 min, persisted throughout the recording periods (up to 110 min after morphine injection, the longest recording time), and were reversed by an opioid receptor antagonist naltrexone. These data suggest that although the overall firing rate of LC neurons was reduced by morphine, the morphine-induced synchronous oscillatory activity may summate temporally and spatially at LC axon terminals and facilitate release of noradrenaline. Noradrenaline is an important neuromodulator and has been shown to induce and facilitate synaptic plasticity at LC target sites. We propose that the morphine-induced long-lasting synchronous oscillatory activity in the LC may be a neuronal signal that could induce synaptic plasticity leading to opioid addiction.