Electrophysiological correlates of presynaptic opiate receptor activation: reduction in norepinephrine-mediated inhibition from the locus coeruleus

Inhibitory responses of rat cerebellar Purkinje cells to locus coeruleus (LC) stimulation and iontophoresis of norepinephrine (NE) were examined before and after administration of morphine to determine whether the inhibitory modulation of NE release by opiates results in a functional impairment in noradrenergic synaptic action. Administration of morphine systemically (0.2-1.2 mg/kg, i.v.) or by iontophoresis reduced inhibitions in Purkinje firing elicited by LC stimulation without affecting depressions in activity induced by postsynaptic applications of NE. This antagonistic effect of morphine on LC-induced inhibition was reversed or prevented by naloxone and mimicked by administration of levorphanol but not dextrorphan. Morphine increased the excitatory response of Purkinje cells to monosynaptic input from the parallel fibers, whereas it blocked gamma-aminobutyric acid-induced inhibitions in firing via a non-opiate receptor-mediated mechanism. These results demonstrate that morphine interferes with synaptic inhibition derived from the LC and suggest that this may occur via activation of presynaptic opiate receptors residing on noradrenergic nerve terminals.     

Electrophysiological evidence against a neurotransmitter role of corticotropin-releasing hormone (CRH) in primary somatosensory cortex

The possible neurotransmitter role of corticotropin-releasing hormone (CRH) was studied in the primary somatosensory cortex of the rat. Electrical activity of single neurones was recorded in layers II–VI of cortex, and in the region of the locus coeruleus. Iontophoresis and pressure ejection were employed to locally apply CRH, and changes in spontaneous, synaptically driven and iontophoretically driven firing were examined. In the cortex, of 62 neurones recorded most (51) were completely unaffected by high and prolonged current/pressure ejections of CRH. Depression of firing was occasionally seen (8 of 62), while a very few (3) were weakly excited. Of 25 cells studied with vibrissal stimulation to evoke excitatory synaptic responses, responses in two cells were depressed and in two they were enhanced. Activity that was evoked by iontophoretic ejection of excitatory amino acids, such as glutamate, was depressed in 6 of 40 cells (none were enhanced). Such effects as were seen were weak and often difficult to reproduce. The effect of CRH on depressions produced by GABA was also tested in four experiments. No effects on the amplitude or duration of the depressions were observed. In contrast recordings made in the midbrain, in the region of the locus coeruleus, resulted in over half the neurones (11 of 20) showing clear reproducible excitatory responses to CRH applications. Solutions used in the experiments were analysed using chromatography, radioimmunoassay and bioassay, and no significant degradation of the peptide was found compared with the synthetic standard (CRH (1–41)). The data provide evidence against CRH acting as a neurotransmitter or modulator in primary cortex, suggesting that the CRH which is localized in certain types of cortical cells is involved in other processes.     

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.     

Effect of Cocaine on Ion Channels and Glutamatergic EPSCs in Noradrenergic Locus Coeruleus Neurons

The locus coeruleus (LC) is an important brainstem area involved in cocaine addiction. However, evidence to elucidate how cocaine modulates the activity of LC neurons remains incomplete. Here, we performed whole recordings in brain slices to evaluate the effects of cocaine on the sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺) channels, and glutamatergic synaptic transmission in the locus coeruleus neurons. Local application of cocaine significantly and reversibly reduced the spontaneous firing rate but did not affect action potential amplitude, rising time, decay time, or half width of noradrenergic locus coeruleus neurons. Moreover, cocaine attenuated the sodium current but did not affect potassium and calcium currents. The N-methyl-d-aspartate receptor mediated excitatory postsynaptic currents were reduced by neuropeptide galanin but not cocaine. All those data demonstrate that cocaine has inhibitory effect on the spontaneous activities and sodium current in locus coeruleus neurons. Therefore, neuromodulation of sodium channel in locus coeruleus neurons may play an important role in drug addiction.     

Effect of deep brain stimulation of GPI on neuronal activity of the thalamic nucleus ventralis oralis in a dystonic patient.

OBJECTIVE: To record the possible effect of acute deep brain stimulation (DBS) of the globus pallidus internus (GPI) on the neuronal activity of the ventralis oralis anterior (VOA) nucleus of the thalamus. METHODS: Under general propofol anaesthesia, extracelullar single unit recordings were performed in VOA of a post-anoxic dystonic patient previously implanted with GPI located electrodes for chronic DBS. RESULTS: Neurons recorded in the VOA could be classified in two cell subpopulations: a high firing rate (16.5 Hz) and low burst index (BI; 15.6) type and a low firing rate (5.5 Hz) and high BI (35.6) type. GPI electrical stimulation reduced the frequency and increased the BI of the high firing rate cells while leaving the other cell type unchanged. CONCLUSION: These results demonstrate that pallidal DBS is able to inhibit a subpopulation of motor thalamic cells and question the pathophysiological model of dystonia based on a low firing rate of GPI cells.     

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.     

Locus coeruleus effects on baroreceptor responsiveness and activity of neurosecretory vasopressin cells

The locus coeruleus (LC) has previously been implicated in the regulation of vasopressin secretion. To further investigate this issue experiments were done in which extracellular recordings were obtained from functionally identified neurosecretory vasopressin (VP) cells of the rat supraoptic nucleus. Electrolytic lesions of the ipsilateral LC reduced the proportion of VP cells inhibited by carotid baroreceptor activation from 93% to 35%; the inhibitory effect of aortic depressor nerve stimulation was unchanged. Electrical stimulation of the LC altered the discharge probability of 20% of VP cells tested, the predominant effect being excitation. In contrast to the effects of electrolytic lesions and electrical stimulation, neither chemical inhibition nor stimulation of the LC, by local injection of neuroactive amino acids, altered VP cell baroreceptor responsiveness or spontaneous discharge. These data indicate that while fibres of passage in the LC region can influence VP cell excitability, particularly responses to carotid baroreceptor activation, LC cells do not regulate VP cell function or, by implication, the secretion of this vasoactive and antidiuretic hormone.     

Electrophysiological study of the response of medial prefrontal cortex neurons to stimulation of the basolateral nucleus of the amygdala in the rat.

The neural connections from the basolateral nucleus of the amygdala (BLA) to the medial prefrontal cortex (MPC) in urethane-anesthetized rats were investigated. Extracellular recordings were made from 200 neurons with spontaneous firing in the MPC, and the BLA was electrically stimulated. The most frequent response to BLA stimulation was inhibition (63.5%). Excitatory responses were found in 17 units (8.5%), while 56 neurons (28%) did not change their spontaneous firing after BLA stimulation. Inhibitory responses showed a wide range of latencies, suggesting the coexistence of mono- and polysynaptic pathways. On the contrary, the excitatory responses seem to be mediated by a monosynaptic pathway. BLA projections to the MPC play a predominantly inhibitory role in the spontaneous activity of prefrontal neurons. This inhibition may modulate central motor systems and motivated behaviors.     

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.     

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.     

Noradrenaline-like terminals in the cat nucleus ventralis posterior of the thalamus

Noradrenaline-like immunoreactivity in the cat nucleus ventralis posterior of the thalamus was investigated using an indirect immunocytochemical technique. Specific antinoradrenaline antibodies, raised in rabbits, were used. It was first verified that these antibodies recognize noradrenaline cells bodies of the locus coeruleus and their ascending axons in the ascending noradrenergic tract. In the nucleus ventralis posterior itself, noradrenaline-like fibers were observed. They were either randomly distributed or grouped around nonlabeled cell bodies. These neurons were generally oblong and measured 60-80 microns. With electron microscopy, preliminary results showed immunoreactive fibers in close apposition to unlabeled cell bodies or dendrites. The precise nature of these profiles was sometimes difficult to ascertain, since experiments were done in presence of detergent. In some cases symmetric synapses might be observed between immunoreactive axon terminals and unlabeled dendrites. The specificity of the reaction is discussed in the light of several control experiments.     

Membrane excitability changes in hindlimb motoneurons induced by stimulation of the locus coeruleus in cats

The present analysis describes the cellular mechanisms underlying the heightened membrane excitability of hindlimb flexor and extensor motoneurons upon stimulation of the locus coeruleus (LC) in unanesthetized, decerebrate cats. In a total of 73 cells, brief train stimuli to the LC at 50-300 microA intensity evoked one of 4 patterns of motoneuronal responses: a simple excitatory postsynaptic potential (EPSP) with weak trailing depolarization, a double-peak EPSP, an EPSP succeeded by a weak hyperpolarization, or a slow rising EPSP. As the initial dominant EPSP was a consistent finding among all cells and the ensuing potentials were variable in polarity, quantitative characterization was focused on the initial EPSP only. In all cells tested (n = 11), the LC-EPSP was accompanied by a decrease in input resistance. The excitatory LC action was further demonstrated by the consistent (n = 25 cells) motoneuron rheobase decrease when the latter was measured coincident with the summit of an LC-EPSP. Furthermore, the time course of the single-spike afterhyperpolarization became shortened during the LC conditioning stimuli (n = 16 motoneurons). Our data show that the descending LC action on motoneurons is typified by an EPSP accompanied by a net decrease in input resistance as well as a concurrent increase in motoneuron electrical excitability.     

Modified coeruleo-cortical noradrenergic neurotransmission after serotonin depletion by PCPA: electrophysiological studies in the rat

To detect eventual modifications in the efficacy of the noradrenergic (NA) coeruleo-cortical system after serotonin (5-HT) depletion by parachlorophenylalanine (PCPA), three electrophysiological parameters were investigated in urethane-anesthetized rats which were treated for 2 days with daily injections of this inhibitor of 5-HT synthesis. 1) The spontaneous activity of locus coeruleus (LC) noradrenergic neurons showed a significant increase in PCPA-treated compared to control rats (4.3 vs. 2.6 Hz). 2) The sensitivity of NA autoreceptors was measured in the LC by the effect of intravenous administrations of clonidine or microiontophoretic applications of NA on spontaneous neuronal firing. In treated rats, clonidine and NA induced a lesser reduction of LC neuron firing than in the controls (27 vs. 75% decreases and 1,367 vs. 280 nC, respectively). 3) The responsiveness of cortical neurons to electrical stimulation of the LC was assessed by peristimulus time histograms in the dorsal fronto-parietal cortex. Following stimulation at 2 or 4 Hz, a majority of spontaneously firing cortical units was inhibited by electrical stimulation of the LC, but the percentage of such units was reduced and showed a decreased responsiveness after PCPA treatment. These findings suggest that following 5-HT depletion by PCPA, cortical NA neurotransmission is markedly reduced in its efficacy in spite of some increase in the spontaneous activity of coeruleo-cortical NA neurons.     

The action of adenosine on noradrenergic neuronal inhibition induced by stimulation of locus coeruleus

Stimulation of locus coeruleus (LC) has been used to induce a noradrenergic inhibition of neurones in the rat cerebral cortex. The local application of adenosine or adenosine monophosphate by microiontophoresis in the cortex was found to produce a reduction of the evoked inhibition if the purine application was begun 20 sec before LC stimulation, but an enhancement of the inhibition if applied up to 35 sec after the LC stimulation. GABA increased the duration of LC inhibition irrespective of time of application. Adenosine and GABA showed no mutual potentiation, but norepinephrine increased the size of responses to both iontophoretic adenosine and GABA. The adenosine-norepinephrine interaction was synergistic, irrespective of the order of application. It is concluded that adenosine may act both presynaptically to inhibit, and postsynaptically to enhance the effects of noradrenergic neurone activation, the dominant effect observed depending on the temporal relationship between LC activation and adenosine application.     

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.     

Differential effects of norepinephrine on hippocampal complex-spike and theta-neurons

The central noradrenergic system has long been postulated to modulate learning and memory. A brain structure known to be important in these functions is the hippocampus. Since the hippocampus receives a noradrenergic projection from the locus coeruleus, knowledge of norepinephrine's actions in the hippocampus may help determine its role in learning and memory. In the present study, the effects of norepinephrine were examined on two hippocampal cell types: complex-spike and theta-neurons. In the hippocampus, there is good evidence that complex-spike cells are pyramidal neurons, while theta-neurons are interneurons. Extracellular action potentials from hippocampal neurons were recorded using multibarrel glass micropipettes. Drugs were locally applied using pressure micro-ejection. Norepinephrine inhibited the spontaneous firing of complex-spike cells, while theta-neurons were excited. The inhibitory response of complex-spike neurons was mediated by an alpha 1-receptor. However, selective agonists for the alpha 2- and beta-noradrenergic receptors excited the complex-spike cells. The noradrenergic-induced excitatory response of theta-neurons was also mediated by alpha 2- and beta-receptors. This study provides evidence that locally applied norepinephrine produces different responses on two types of hippocampal neurons. Furthermore, these differential responses arise primarily from the activation of distinct populations of noradrenergic receptors.     

Noradrenergic actions of purkinje and locus coeruleus neurons in culture

1. Explant cultures prepared from neonatal mice were used to study the actions of ionto-phoretically applied noradrenaline (NA) on Purkinje and locus coeruleus neurons, with extracellular and intracellular recording respectively.

2. NA depressed spontaneous activity of Purkinje neurons and enhanced excitatory responses to glutamate in 14/16 cells.

3. In cultures older than 26 days, NA hyperpolarized LC neurons but had no effect on or depressed depolarizing responses to glutamate. The hyperpolarizations were blocked by the selective ₂ antagonist yohimbine.

4. Enhancement of glutamate responses is not a ubiquitous characteristic of NA action and appears not to be associated with ₂ adrenergic receptors.     

Spontaneous and auditory-evoked activity of medial agranular cortex as a function of arousal state in the freely moving rat: interaction with locus coeruleus activity

To characterize the electrophysiological properties of neurons in the medial agranular frontal cortex (Fr2) with respect to arousal level and locus coeruleus (LC) activity, we recorded spontaneous and auditory-evoked single unit activity in these areas simultaneously during different states of arousal in the rat. In the low-arousal state, as determined by EEG, 14/56 Fr2 neurons showed a tonic increase in spontaneous firing rate and 9/56 presented a clear inhibitory response to tone (onset latency 37 ms, duration 200 ms). The inhibitory response was not clear during the high-arousal state. Cross-correlation analysis of pairs of Fr2 and LC units, excluding activity during the actual tone, also showed a negative correlation from 120 ms before, to 170 ms after, Fr2 discharge in 5/63 pairs, only during low arousal. Significantly, 4/5 of the Fr2 neurons having this negative correlation with LC were included in that population which showed a tonic increase in spontaneous firing rate and inhibited to tone during low arousal. LC neurons, on the other hand, all showed excitation to tone stimulation (peak latency 30 ms), and response amplitude was not affected by changes in arousal level. The negative correlation in spontaneous activity, as well as their differential responses to tone, suggests an interaction between a select population of Fr2 neurons and the LC during the low-arousal state. Future studies with lesion or pharmacological manipulations would be necessary to confirm the presence of this interaction.     

Entopeduncular lesions facilitate and thalamic lesions depress spontaneous and drug-evoked motor behavior in the hemiparkinsonian rat

Pallidotomy is a neurosurgical procedure designed to ameliorate the akinesia and bradykinesia associated with Parkinson's disease. In the present study, the effects of pallidal-like lesions on motor behavior in the hemiparkinsonian rat were compared to the effects of lesions in the ventrolateral thalamus, a target of entopeduncular projections feeding motor-related information to motor cortex. Six aspects of spontaneous and evoked behavior induced by amphetamine and apomorphine in the hemiparkinsonian rat with either bilateral electrolytic entopeduncular lesions or bilateral electrolytic ventrolateral thalamic lesions were measured for 60 min. Saline or amphetamine, 5 mg/kg, or apomorphine, 0.3 mg/kg, were administered IP 5 min before the tests. The results show that on all measures except time spent resting the hemiparkinsonian rats with the entopeduncular lesions were more active than the hemiparkinsonian rats with the thalamic lesions. The asymmetrical rotation responses to dopamine receptor stimulation evoked by amphetamine and apomorphine were influenced by the general effect on gross motor behavior, as shown by the response being very large in the entopeduncular group and very small in the thalamic group. These results are consistent with current thinking about the functional organization of the basal ganglia according to which damage of the entopeduncular nucleus reduces its inhibitory control of the thalamic motor regions, thereby promoting thalamic facilitation of motor cortex, and damage to the thalamic motor regions has the opposite effect. These effects of the lesions translate, respectively, into hyperactivity and hypoactivity without blocking the asymmetrical rotation response of the hemiparkinsonian rat.     

Subsensitivity of catecholaminergic neurons to direct acting agonists after single or repeated electroconvulsive shock

Spontaneous firing rates and changes in firing rate in response to an intravenously administered dose of apomorphine were measured after various electroconvulsive shock (ECS) treatment regimens from dopaminergic cells of the substantia nigra in urethane-anesthetized rats. Similar measurements were obtained from noradrenergic neurons of the locus coeruleus before and after intravenous injection of clonidine. A significant decrement in the inhibition of spontaneous firing in response to intravenous administration of these agonists was observed following multiple or single ECS treatment in both substantia nigra and locus coeruleus cells. There was a consistent but nonsignificant tendency for cells in both areas of the brain from treated animals to display higher rates of spontaneous firing than their respective sham-shocked controls. Both the effects on base-line rates of spontaneous activity and on the depression of firing rate in response to drug administration were found to be independent of repeated treatment. A significant negative correlation was obtained between base-line firing rate and percentage depression to the autoreceptor agonist, but this correlation alone was insufficient to account for the observed differences in the drug response. These results are discussed with respect to possible mechanisms of action of electroconvulsive therapy in the treatment of depression.