Locus coeruleus stimulation potentiates local inhibitory processes in rat cerebellum

We previously reported that a low threshold action of norepinephrine (NE) on the cerebellar circuitry is expressed as an amplification of the inhibitory action of gamma aminobutyric acid (GABA) on Purkinje cell activity. Here we examined the effects of locus coeruleus (LC) stimulation on “off-beam” inhibitions of Purkinje cell firing induced by activation of local basket and stellate cell interneurons to determine whether endogenous NE, released from synaptic terminals, could induce a comparable enhancement of GABA-mediated synaptic input to these neurons. Stimulation of LC, at current intensities which by themselves were subthreshold for directly affecting background activity of Purkinje neurons, markedly increased off-beam inhibitory neuronal responses. Iontophoretic application of the beta-adrenergic blocker sotalol reversibly antagonized this enhancement of synaptic inhibition. In comparison, the potentiative effects observed with LC stimulation were increased by iontophoresis of the alpha-adrenergic blocker phentolamine. LC -induced increases in off-beam inhibition were not observed after destruction of cerebellar noradrenergic terminals by 6-hydroxydopamine. These results suggest that noradrenergic input from the LC can augment the efficacy of conventional GABA-mediated inputs synapsing on the Purkinje cell.     

Locus coeruleus stimulation potentiates Purkinje cell responses to afferent input: The climbing fiber system

In cerebellum, the evoked responses of the Purkinje cell to both excitatory and inhibitory afferent input have previously been shown to be enhanced by local iontophoresis of norepinephrine (NE). The influence of locus coeruleus (LC) conditioning stimulation on Purkinje cell responses to climbing fiber input was examined to determine whether endogenous NE, released from synaptic terminals, could exert similar potentiative effects. Stimulation of LC, at intensities which by themselves were subthreshold for directly affecting background activity, markedly enhanced complex spike excitation of Purkinje cells elicited by activation of climbing fiber inputs from sensorimotor cortex. Depressant responses observed after complex spike excitation were also augmented by the LC conditioning. Iontophoretic application of sotalol, a specific β-adrenergic receptor blocker, reversibly antagonized this facilitation of climbing fiber-evoked responses. In addition, the potentiative effects of LC stimulation were not observed after destruction of NE-containing axons and terminals in cerebellum by 6-OHDA. These results suggest that noradrenergic input from the LC can enhance the efficacy of climbing fiber synaptic action on the Purkinje cell, and are thus consistent with the hypothesis of a ‘modulatory’ role rather than a specific information transfer function for NE in cerebellum.     

Electrophysiological effects of locally applied noradrenergic agents at cerebellar Purkinje neurons: receptor specificity

We have investigated the receptor subtype(s) mediating the noradrenergic inhibition of cerebellar Purkinje cell spontaneous firing rate using local application of specific agonists and antagonists, in situ, via pressure microejection. Extracellular action potentials were recorded from Purkinje neurons in anesthetized Fischer 344 rats. Timolol, a beta-receptor antagonist, did not affect norepinephrine (NE)-induced inhibition in 9 of 12 cells studied. Phentolamine, an alpha-receptor antagonist, blocked the effect of NE in 8 of 11 cells. To further determine the subtype of alpha-receptor involved, the effects of the alpha 1-antagonist prazosin and alpha 2-antagonists idazoxan and yohimbine were examined. While prazosin had no effect on NE-mediated inhibition, both idazoxan and yohimbine blocked NE effects. Idazoxan was also successful in blocking phencyclidine (PCP), an indirect noradrenergic agonist. The inhibitory action of NE upon Purkinje cell firing rate was mimicked by the selective alpha 2-agonist clonidine; this action of clonidine was blocked by idazoxan but not by timolol or prazosin. In addition, the alpha 1-adrenergic agonist phenylephrine and the beta-adrenergic agonist isoproterenol inhibited Purkinje cell firing rate. Phenylephrine effects were blocked by prazosin but not by timolol or idazoxan. Isoproterenol-induced inhibition was blocked by timolol but not phentolamine. Taken together, these studies suggest that both alpha- and beta-receptors alter Purkinje cell firing rate; the depressant action of locally applied NE, however, seems to be mediated primarily via an alpha 2-adrenergic receptor.     

Interactions of norepinephrine with Purkinje cell responses to putative amino acid neurotransmitters applied by microiontophoresis

In cerebellum, the evoked responses of the Purkinje neuron to both excitatory and inhibitory afferent input are enhanced by local norepinephrine (NE) administration. To determine the nature of this synergistic interaction between NE and the synaptic inputs, Purkinje cell responses to microiontophoretically applied amino acid neurotransmitters were examined before, during, and after NE iontophoresis. NE was found to preferentially augment GABA-induced inhibition, whereas it antagonized inhibition produced by glycine. This enhancement of GABA inhibition was seen at NE doses which caused minimal change in spontaneous activity, and at times after spontaneous discharge returned to control levels following NE application. Dopamine did not facilitate the response to GABA even at doses having direct depressant effects on spontaneous discharge. Glutamate-evoked excitations and subsquent depressions were also augmented during NE administration, relative to the level of background activity. Thus, NE selectively enhanced responses of Purkinje neurons produced by microiontophoretic application of amino acids postulated to be cerebellar neurotransmitters, supporting the hypothesis that NE acts on postsynaptic processes to increase the responsiveness of the Purkinje cell to afferent input. These data also provide evidence supporting the concept that a primary effect of noradrenergic input to cerebellum may be to modulate the action of other transmitters.     

Potentiation of GABA inhibitory action in cerebellum by locus coeruleus stimulation

In cerebellum, excitatory and inhibitory responses of Purkinje cells, produced both synaptically and by microiontophoresis of putative amino acid neurotransmitters, have been shown previously to be enhanced during NE iontophoresis. The influence of locus coeruleus conditioning stimulation on Purkinje cell responses to GABA iontophoresis was examined to determine whether endogenous NE, released from synaptic terminals, could exert similar modulatory effects. Locus coeruleus stimulation at current intensities which alone elicited no direct depression of Purkinje cell spontaneous discharge potentiated the inhibition produced by GABA. Iontophoretic application of sotalol, a specific β-adrenergic blocker, antagonized this enhancement of GABA inhibition. Repetitive activation of the classic non-adrenergic cerebellar afferents did not enhance the GABA response, despite causing a direct depression in spontaneous rate. A neuromodulatory role is suggested for tonic adrenergic input in the mammalian central nervous system.     

Electrically-evoked release of norepinephrine in the rat cerebellum: an in vivo electrochemical and electrophysiological study.

Norepinephrine (NE) release from the locus coeruleus (LC) afferents to the cerebellar cortex of urethane anesthetized rats was achieved by electrical stimulation of the locus coeruleus or by local administration of potassium into the cerebellum. Both methods evoked an overflow of NE-like electroactive species. Electrically-evoked and potassium-induced overflow of NE-like responses were found to be reversible and reproducible. Releases were not observed in cerebellar white matter, an area which is relatively devoid of monoamine containing terminals. Systemic administration of desipramine, a potent and selective norepinephrine re-uptake blocker, significantly augmented the electrically-evoked electrochemical responses. Measurements of evoked release taken using high-speed chronoamperometry support the idea that a predominant contributor to electrically-induced signals was NE. Electrophysiological recordings of single Purkinje cells were performed with the same Nafion-coated single carbon fiber electrodes used for electrochemical recordings. Electrical stimulation of the LC was seen to depress Purkinje cell firing rates; an increase in electroactive species was detected at the same site that paralleled the time course of the electrophysiological response. These studies provide further direct evidence that the LC norepinephrine-containing cells have a direct inhibitory effect on Purkinje cells in the cerebellum, and that both pre- and postsynaptic events can be measured with the same recording sensor.     

Activation of lateral geniculate neurons by locus coeruleus or dorsal noradrenergic bundle stimulation: Selective blockade by the alpha1-adrenoceptor antagonist prazosin

Presentation of a stimulus train to the locus coeruleus (LC) or dorsal noradrenergic bundle (DB) resulted in a facilitation of thev spontaneous firing of single units in the dorsal lateral geniculate nucleus (LGNd) of the rat. These stimulation effects were blocked by the alpha₁-adrenoceptor antagonists WB-4101 and prazosin. Both drugs also blocked the activation of LGNd neurons by iontophoretic norepinephrine (NE). The cholinergic agonists acetylcholine (ACh) and carbachol (CCh) activated LGNd neurons in a similar fashion to NE, however, these responses were selectively blocked by the muscarinic antagonist scopolamine. The response to ACh was also sensitive to WB-4101 suggesting that the drug possesses some cholinergic blocking activity. In contrast to WB-4101, prazosin displayed a high degree of selectivity for noradrenergic but not cholinergic response. On the basis of the observation that prazosin selectively antagonizes both the stimulation effects and iontophoretic NE (but not CCh), we conclude that activation of LGNd neurons by LC or DB stimulation is mediated predominantly via the release of NE from coeruleo-geniculate fibers, rather than the inadvertent activation of a cholinergic pathway. Moreover, inasmuch as the systemic administration of prazosin effectively blocks central noradrenergic neurotransmission at dosescomparable to those uses clinically, the possibility that prazosin exerts its antihypertensive action in part via a central mechanism requires further investigation.     

Effects of dopamine and norepinephrine on neuronal activity of the olfactory tubercle

The effects of iontophoretic administration of norepinephrine (NE) and dopamine (DA) on olfactory tubercle (OT) neurons that respond to lateral hypothalamus (LH) or locus coeruleus (LC) electrical stimulation were studied. NE and DA decreased the frequency of OT neurons which were increased or decreased by the LH stimulation. An increased firing of OT neurons following NE or DA administration was less frequently observed. NE administration decreased the firing of OT neurons that responded to LC stimulation. These results suggest that the LC fibers which reach the OT use NE as a neurotransmitter. DA administration also suppressed the unitary discharge of OT neurons responding to LC stimulation. The increase in frequency of OT neurons observed following LH stimulation cannot be attributed to DA. The possibility that other suspected neural transmitters are involved in this effect is discussed.     

Electrophysiology of Purkinje neurons in the weaver mouse: iontophoresis of neurotransmitters and cyclic nucleotides, and stimulation of the nucleus locus coeruleus.

We compared the Purkinje cells of adult normal and weaver mutant (wv/wv) mice by iontophoretic and electrophysiological tests. Although weaver Purkinje cells fire spontaneously at a rate (38 Hz) similar to normal mouse neurons (40 Hz), several abnormalities of firing were seen: high frequency bursts of single (simple) spikes occurred in 5-10-sec episodes in 38% of weaver cells, compared to 8% in normal mice; spontaneous complex spikes (climbing fiber-like burst responses) occurred in several different forms in a given Purkinje cell. As in normal mice and rats, the spontaneous single spike activity is readily depressed by electrical stimulation of the locus coeruleus, the presumed source of a dense noradrenergic plexus in the weaver cerebellar cortex. In a preliminary experiment the adrenergic blocking agent, fluphenazine, antagonized the responses to locus coeruleus stimulation. Iontophoresis of norepinephrine (NE), GABA and serotonin (5-HT) also uniformly depressed Purkinje cell single spike activity in all normal and weaver mice; cyclic AMP depressed 55% of normal and 70% of weaver Purkinje cells. Glutamate was always excitatory. The only qualitative difference was seen with acetylcholine, which was mostly inhibitory in normal mouse, but increased the firing rate in 42% of weaver Purkinje neurons. Cyclic GMP was predominantly excitatory in both types. Thus, despite the absence of parallel fibers, weaver Purkinje neurons grossly resemble normal Purkinje cells electrophysiologically as well as morphologically. Since several sites of indirect presynaptic actions are eliminated in weaver, our results further substantiate the direct post-synaptic inhibitory nature of GABA, 5-HT and NE, and the noradrenergic pathway from locus coeruleus to Purkinje cells. Similarly, consistent inhibitory responses to cyclic AMP in the weaver support the previously hypothesized role of cyclic AMP in the post-synaptic inhibitory response to NE.     

Pyramidal neurons in rat prefrontal cortex show a complex synaptic response to single electrical stimulation of the locus coeruleus region: Evidence for antidromic activation and GABAergic inhibition using in vivo intracellular recording and electron microscopy

Cognition and acquisition of novel motor skills and responses to emotional stimuli are thought to involve complex networking between pyramidal and local GABAergic neurons in the prefrontal cortex. There is increasing evidence for the involvement of cortical norepinephrine (NE) deriving from the nucleus locus coeruleus (LC) in these processes, with possible reciprocal influence via descending projections from the prefrontal cortex to the region of the LC. We used in vivo intracellular recording in rat prefrontal cortex to determine the synaptic responses of individual neurons to single electrical stimulation of the mesencephalic region including the nucleus LC. The most common response consisted of a late-IPSP alone or preceded by an EPSP. The presence of an early-IPSP following the EPSP was sometimes detected. Analysis of the voltage dependence revealed that the late-IPSP and early-IPSP were putative K⁺- and Cl⁻ dependent, respectively. Synaptic events occurred following short delays and were inconsistent with the previously reported time for electrical activation of unmyelinated LC fibers. Moreover, systemic injection of the adrenergic antagonists propranolol (β receptors), or prazosin (α1 receptors), did not block synaptic responses to stimulation of the LC region. Finally, certain neurons were antidromically activated following electrical stimulation of this region of the dorsal pontine tegmentum. Taken together, these results suggest that the complex synaptic events in pyramidal neurons of the prefrontal cortex that are elicited by single electrical stimulation of the LC area are mainly due to antidromic activation of cortical efferents. Further insight into the chemical circuitry underlying these complex synaptic responses was provided by electron microscopic immunocytochemical analysis of the relations between the physiologically characterized neurons and either 1) GABA or 2) dopamine-β-hydroxylase (DBH), a marker for noradrenergic terminals. GABA-immunoreactive terminals formed numerous direct symmetric synapses on somata and dendrites of pyramidal cells recorded and filled with lucifer yellow (LY). In contrast, in single sections, noradrenergic terminals immunoreactive for DBH rarely contacted LY-filled somata and dendrites. These results support the conclusion that IPSPs observed following single electrical stimulation of the LC region are mediated bu GABA, with little involvement of NE. These IPSPs, arising from antidromic invasion of mPFC cells innervating the LC, may improve the signal-to-noise ratio and favor a better responsiveness of neighboring neurons to NE released in the mPFC. © 1996 Wiley-Liss, Inc.     

Electrophysiological actions of norepinephrine in rat lateral hypothalamus. I. Norepinephrine-induced modulation of LH neuronal responsiveness to afferent synaptic inputs and putative neurotransmitters

The present studies were conducted as part of an ongoing investigation of the effects of norepinephrine (NE) in neuronal circuits of the mammalian brain. In this report, we describe noradrenergic actions in the lateral hypothalamus (LH), an area which has been implicated in the central integration of cardiovascular regulatory mechanisms, fluid balance and ingestive behaviors. Microiontophoretically applied NE was interacted with extracellularly recorded responses of LH neurons to iontophoretically applied putative neurotransmitters gamma-aminobutyric acid (GABA), acetylcholine (ACh) and glutamate (Glu); and activation of known input pathways from the reticular thalamus (RT) and the lateral preoptic area (LPO). Peri-event histograms of cell responses were computed before, during and after NE microiontophoresis (5-50 nA) and used to quantitatively evaluate monoamine-induced effects on spontaneous and stimulus evoked activity of LH neurons. In 16 of 23 LH neurons, RT-stimulus-induced inhibition was markedly prolonged from a mean of 28.3 +/- 4.8 ms to 44.7 +/- 5.2 ms, during iontophoretic application of NE. In 22 of 38 LH cells, LPO-stimulus-induced excitatory responses were enhanced above control levels during NE administration. In further tests, inhibitory responses of LH cells to iontophoretic pulses of GABA were potentiated during NE administration in 69% (24 of 35) of the cases tested. ACh-induced excitation was potentiated in 9 of 21 cells. In 4 of these cases, otherwise subthreshold doses of ACh caused marked increases in cell firing during the period of NE administration. By contrast, Glu-evoked excitation was antagonized by NE iontophoresis in 65.5% (17 of 26) of LH cells tested. These findings indicate that, as in other noradrenergic target regions of the CNS, NE can facilitate synaptically mediated responses of LH neurons. Taken together these observations suggest that NE may play an important regulatory role in the synaptic transfer of information within LH circuits, and consequently exert considerable influence over the homeostatic functions mediated by this structure.     

Modification of the visual response properties of cerebellar neurons by norepinephrine

Extracellular recordings were conducted in the paraflocculus of anesthetized Long-Evans pigmented rats to determine how ionotophoresis of norepinephrine (NE) affects the responsiveness of individual Purkinje cells and interneurons to presentations of visual stimuli within their visual receptive fields. Presentations of moving or stationary visual stimuli during the control (pre-NE) period elicited simple spike excitations or inhibitory responses in slightly more than one-half (55%, n = 32) of the cells tested (20 of 38 Purkinje cells, 12 of 20 interneurons). The predominant effect of NE iontophoresis was to improve visually evoked responses in those neurons which showed modulations in their simple spike discharge to control presentations of visual stimuli. A clear enhancement of visual responses by NE (i.e., absolute increase over control) was observed in 18 of the units, and in 12 of the 14 remaining cells, reductions in stimulus-bound discharge during catecholamine iontophoresis were accompanied by much larger depressions in background activity, resulting in a net enhancement in the ratio of signal-to-noise. NE differentially affected responses to stimulus movement in the preferred and non-preferred direction in one-third of these neurons, such that directional selectivity was increased. However, the orientation bias of individual units was unchanged by NE. Iontophoretic application of the beta-adrenergic antagonist sotalol but not the alpha-adrenergic antagonist phentolamine blocked these facilitating noradrenergic effects. An additional feature of noradrenergic action was revealed in tests conducted in 26 cells which did not respond to control presentations of visual stimuli. Iontophoresis of NE resulted in the elicitation of visual responses in 11 of these units, suggesting the possibility that NE might act in some cases to gate the efficacy of subliminal synaptic input conveyed by classical afferent channels. It is proposed that an important aspect of noradrenergic action within local cerebellar circuits might be to refine the receptive field properties of individual neuronal elements and thereby improve information flow through the cerebellum.     

Comparative effects of sciatic nerve stimulation, blood pressure, and morphine on the activity of A5 and A6 pontine noradrenergic neurons

The changes in the firing rate of A5 and A6 (locus coeruleus, LC) noradrenergic neurons induced by sciatic nerve stimulation, norepinephrine-induced elevations of blood pressure (BP) and systemic administration of morphine were studied in rats anesthetized with urethane, paralyzed and ventilated. Stimulation of the contralateral sciatic nerve with single shocks of low intensity (0.2 ms, 0.5 mA) produced a strong excitation of LC neurons with a latency of 14-18 ms. By contrast, shocks of similar intensities were ineffective in driving A5 cells. Higher stimulus intensities produced a low efficacy driving of A5 cells with a very long latency (120-250 ms). The efficacy of the stimulation could be increased by delivering trains of 4-8 stimuli but the latency remained very long. Norepinephrine-induced elevation of arterial pressure (140-160 mm Hg range) silenced the vast majority of A5 neurons. By contrast, the effects of blood pressure on locus coeruleus cells were of small amplitude, poorly reproducible and their time course was generally not correlated with the blood pressure alterations. Finally, the administration of 5 mg/kg of morphine i.v. silenced virtually all LC neurons while the majority of A5 cells were excited by the drug. These results provide evidence for the existence of a differential innervation of the two groups of pontine noradrenergic neurons investigated.     

Interaction of norepinephrine with cerebrocortical activity evoked by stimulation of somatosensory afferent pathways in the rat

This study investigated the action of norepinephrine (NE) on transmission of information through somatosensory cortical neuronal circuits. In the forelimb region of rat somatosensory cortex (SI), single-unit responses to natural stimulation (foot tap) of afferent pathways were recorded before, during, and after microiontophoretic application of NE. Actions of NE were quantitatively assessed by computer-based analysis of poststimulus time histograms. Overall, NE was found to enhance both excitatory and inhibitory responses generated by the afferent synaptic input. In 78% of the cells tested, low doses of NE differentially suppressed background discharge more than stimulus-bound excitation such that the signal to noise ratio was enhanced approximately twofold. Evoked spiking in 12 cells was quantitatively increased above control values during NE administration. In 82% of the units examined, NE augmented stimulus-bound inhibition and postexcitatory suppression of activity. Potentiation of inhibition was observed in 5 cells at doses of NE which caused little or no depression of spontaneous activity. These observed effects on neuronal responsiveness to afferent synaptic input often persisted for several minutes after termination of NE iontophoresis. Such modulatory actions of NE were demonstrated for cells situated throughout the vertical extent of the cortex. These results suggest that low amounts of NE may facilitate transfer of afferent information within the cerebral cortical circuitry and are consistent with a modulatory role rather than a specific information transfer function for NE.     

Noradrenergic modulation of somatosensory cortical neuronal responses to lontophoretically applied putative neurotransmitters

We examined the interaction of norepinephrine (NE) applied iontophoretically in small doses with the responsiveness of somatosensory cortical neurons to the putative neurotransmitter substances acetylcholine (ACh) and gamma-aminobutyric acid (GABA). Neuronal responses to microiontophoretic pulses (8- to 10-s duration at 45-s intervals) of ACh and GABA were examined before, during, and after NE iontophoresis. Computer-generated histograms used for quantitation of drug responses revealed a NE-induced enhancement of neuronal responsiveness to both ACh and GABA. NE differentially suppressed the spontaneous firing rate more than activity during ACh-induced excitation such that the excitatory response was enhanced relative to background discharge in 86% of the cells tested. In 13 of 35 cells tested, ACh responses were potentiated above control values. GABA-induced inhibition of cortical neuron spontaneous discharge was augmented during iontophoretic application of NE in 94% of the cells examined. Dopamine, even at doses sufficient to depress background firing rate, was not effective in facilitating responses to either ACh or GABA. These results are consistent with the hypothesis that a primary function of the central noradrenergic system is to enhance the efficacy of postsynaptically acting neurotransmitters.     

Cannabinoids modulate spontaneous neuronal activity and evoked inhibition of locus coeruleus noradrenergic neurons

The noradrenergic pathway arising from the locus coeruleus (LC) is involved in the regulation of attention, arousal, cognitive processes and sleep. These physiological activities are affected by Cannabis exposure - both in humans and laboratory animals. In addition, exogenous cannabinoids, as well as pharmacological and genetic manipulation of the endocannabinoid system, are known to influence emotional states (e.g. anxiety) for which a contributory role of the LC-noradrenergic system has long been postulated. However, whether cannabinoid administration would affect the LC neuronal activity in vivo is still unknown. To this end, single-unit extracellular recordings were performed from LC noradrenergic cells in anaesthetized rats. Intravenous injection of both the synthetic cannabinoid agonist, WIN55212-2, and the main psychoactive principle of Cannabis, Delta9-tetrahydrocannabinol, dose-dependently increased the firing rate of LC noradrenergic neurons, with WIN55212-2 being the most efficacious. Similar results were obtained by the administration of these drugs into a lateral ventricle. Cannabinoid-induced stimulation of LC noradrenergic neuronal activity was counteracted by SR141716A, a cannabinoid receptor antagonist/reverse agonist, which by itself slightly reduced LC discharge rate. Moreover, WIN55212-2 suppressed the inhibition of noradrenergic cells produced by stimulation of the major gamma-aminobutyric acid (GABA)ergic afferent to the LC, the nucleus prepositus hypoglossi. Altogether, these findings suggest the involvement of noradrenergic pathways in some consequences of Cannabis intake (e.g. cognitive and attention deficits, anxiety reactions), as well as a role for cannabinoid receptors in basic brain activities sustaining arousal and emotional states.     

Opposing effects of morphine and the neurotrophins, NT-3, NT-4, and BDNF, on locus coeruleus neurons in vitro

The development of noradrenergic locus coeruleus (LC) neurons is subject to regulation by multiple epigenetic signals. To examine the potential regulation of LC ontogeny by opiates and neurotrophins, we studied the effects of morphine and NT-3, NT-4, and BDNF on the survival and differentiation of LC neurons from prenatal rats in dissociated cell culture. Noradrenergic cells were identified and counted following tyrosine hydroxylase (TH) immunocytochemistry, and their state of differentiation was assessed by measuring norepinephrine (NE) uptake. Treating LC cultures with morphine starting on day 1 after plating resulted in a 20% decrease in NE uptake and a small (12%) but significant decrease in the number of TH-immunoreactive (TH + ) cells. Application of morphine on day 4 after plating had the same effect on NE uptake without influencing TH + cell number. This effect of morphine was blocked by concomitant exposure to naloxone (an opioid receptor antagonist), and mimicked by exposure to opioid peptides. Treatment of cultures with the neurotrophins, NT-3 or NT-4, increased NE uptake and TH + cell number, as reported previously. Moreover, we show for the first time that brain-derived neurotrophic factor (BDNF) exerts similar effects, with a large (110%) increase in NE uptake and a modest (20%) increase in TH + cell number. Cotreatment of LC cultures with morphine and NT-3 resulted in an attenuation of the NT-3 effect on both NE uptake and the number of TH + cells. In contrast, cotreatment of LC cultures with morphine and NT-4 or BDNF attenuated the neurotrophin effect on TH + cell number but not on NE uptake. Our results raise the possibility that opioid peptides may modulate the influence of neurotrophins on LC neuronal survival and differentiation.     

GABA-mediated inhibition of locus coeruleus from the dorsomedial rostral medulla

Recent anatomic studies in our laboratory (Aston-Jones et al., 1986) identified the nucleus prepositus hypoglossi (PrH) in the dorsomedial medulla as a major afferent of the locus coeruleus (LC). In the present studies, the influence of projections from PrH to LC was assessed in anesthetized rats. Focal electrical stimulation of PrH inhibited the spontaneous discharge of 42 of 47 LC neurons; the latency to onset of such inhibition was 19.8 +/- 2.5 msec and its duration was 172.4 +/- 10.4 msec. PrH-evoked inhibition of LC neurons was unaffected by administration of the opiate receptor antagonist naloxone or the alpha 2-receptor antagonist idazoxan but was substantially reduced by systemic picrotoxin, an antagonist of GABA. The GABAA receptor antagonist bicuculline methiodide blocked the inhibition from PrH, whether applied by local microinfusion or iontophoresis into the LC. These results lead us to propose that PrH provides a direct inhibitory synaptic input to LC, for which GABA is the likely transmitter.     

Discrete local application of corticotropin-releasing factor increases locus coeruleus discharge and extracellular norepinephrine in rat hippocampus.

The most prominent afferents impinging upon the noradrenergic neurons of the locus coeruleus (LC) utilize GABA and glutamate. However, peptide neurotransmitters such as galanin, neuropeptide Y, and corticotropin-releasing factor (CRF) have also been localized to LC afferents. The evidence for CRF modulation of LC activity was examined in the present studies. Specifically, the impact of local CRF administration on both LC-NE discharge characteristics and release of norepinephrine (NE) in hippocampus was determined. First, the ability of CRF microinfused into the LC area to increase NE efflux in the dorsal hippocampus was determined using in vivo microdialysis techniques in awake rats. CRF into the LC dose-dependently increased extracellular NE in the ipsilateral hippocampus. Second, a more detailed analysis was performed in halothane-anesthetized rats by characterizing the electrophysiological activity of LC-NE neurons in response to local application of CRF. Changes in the firing rate and pattern of single LC-NE neurons were measured while simultaneously monitoring the extracellular level of NE in hippocampus. A dose of 30 ng CRF applied directly into LC via pressure ejection elicited an 88% increase in the discharge rate of LC-NE neurons and increased the incidence of burst firing from 14% to 33%. This manipulation simultaneously increased extracellular NE in hippocampus by 63%. The CRF-induced increases in discharge rate of LC-NE neurons and extracellular NE efflux in hippocampus were prevented by prior i.c.v. administration of the CRF antagonist, d-PheCRF(12-41 )(3 microg / 3 microl). The present findings demonstrate that CRF applied directly into the LC increases both the activity of LC-NE neurons and the release of NE in an LC terminal region. The shift in activity of LC-NE neurons to more burst-like firing in response to CRF may provide a means for enhanced release of NE in LC projection fields. This is the first report to demonstrate a dose-dependent increase in extracellular NE levels evoked by intra-LC infusion of CRF in unanesthetized animals.     

Electrophysiological characterization of adrenoceptors in the rat dorsal hippocampus. III. Evidence for the physiological role of terminal alpha 2-adrenergic autoreceptors

The present electrophysiological studies were undertaken to assess the role of terminal alpha 2-adrenergic autoreceptors in regulating noradrenergic synaptic transmission in the rat CNS. The effectiveness of the electrical stimulation of the locus coeruleus (LC) in suppressing the firing activity of pyramidal neurons was determined in the dorsal hippocampus. Intravenous clonidine, an alpha 2-adrenergic agonist, decreased the effectiveness of the LC stimulation, without altering the effect of microiontophoretically applied norepinephrine. The subsequent i.v. administration of low doses of idazoxan, an alpha 2-adrenergic antagonist, reversed this effect of clonidine on the LC stimulation. To ascertain that the effect of clonidine administered i.v. was indeed attributable to its action on noradrenergic terminals, it was applied locally by microiontophoresis; it decreased the effectiveness of the LC stimulation. Another paradigm used to assess the function of terminal alpha 2-adrenoceptors was to increase the frequency of the LC stimulation from 1 to 5 Hz. This resulted in a 5-fold decrease of the effectiveness of the stimulation. That this was attributable to an enhanced activation of terminal alpha 2-adrenoceptors was suggested by the reversal of this effect of increasing the frequency of the LC stimulation by intravenous idazoxan. Furthermore, the degree of enhancement of the effectiveness of the LC stimulation by idazoxan was much greater at 5 than at 1 Hz. These results provide novel electrophysiological evidence for the potent regulatory role of terminal alpha 2-adrenoceptors on noradrenergic neurotransmission.