Mutual interactions among cholinergic, noradrenergic and serotonergic neurons studied by ionophoresis of these transmitters in rat brainstem nuclei

In urethane-anesthetized rats, single neuronal activity was recorded in or around the central gray of the caudal mesencephalon to rostral pons with multibarrel microelectrodes for ionophoretic application of acetylcholine, noradrenaline and serotonin. Neurons were classified by spike shape into broad-spike and brief-spike neurons. In the laterodorsal tegmental nucleus, locus coeruleus or dorsal raphe, broad-spike neurons, marked by Pontamine Sky Blue and discriminated in sections processed for histochemistry of reduced nicotinamide adenine dinucleotide phosphate diaphorase or Nissl staining, were presumed to be cholinergic, noradrenergic or serotonergic, respectively. The majority of these neurons were inhibited through autoreceptors, except some laterodorsal tegmental neurons which might not be furnished by autoreceptors. Noradrenaline and serotonin inhibited more than two-thirds of the laterodorsal tegmental neurons tested, while a few neurons were excited by noradrenaline. Though effects of noradrenaline on dorsal raphe neurons and those of serotonin on locus coeruleus neurons were not clear in many neurons tested, neurons affected in these examinations (30%) were all inhibited clearly and no excitatory effect was observed. Acetylcholine exerted inhibition on about one-half of dorsal raphe neurons, while effects of acetylcholine on locus coeruleus neurons were the only case in the present study in which excitation was the major effect, though more than a half of locus coeruleus neurons were not sensitive to this drug. Thus, in this study some new data on the pharmacological properties of the cholinergic laterodorsal tegmental neurons were obtained. In addition, mutual interactions between brainstem cholinergic, noradrenergic and serotonergic neurons were assayed by comparing the pharmacological properties of these neurons tested with a uniform procedure. The interactions between these diffuse projection neurons may be involved in neural mechanisms controlling vigilance, wakefulness and/or sleep.     

Role of adenosine in behavioral state modulation: a microdialysis study in the freely moving cat

There is considerable evidence to suggest that the activity of forebrain and mesopontine cholinergic neurons is intimately involved in electroencephalographic arousal. Furthermore, our previous in vitro investigation suggested that both cholinergic systems are under a powerful tonic inhibitory control by endogenous adenosine. We thus examined the in vivo effect, on electrographically defined behavioral states, of microdialysis perfusion of adenosine into the cholinergic zones of the substantia innominata of the basal forebrain and the laterodorsal tegmental nucleus of freely moving cats. Localized perfusion of adenosine into either the basal forebrain or the laterodorsal tegmental nucleus caused a marked alteration in sleep-wake architecture. Adenosine (300 microM) perfused into either the basal forebrain or laterodorsal tegmental nucleus produced a dramatic decrease in waking, to about 50% of the basal level. Perfusion into the basal forebrain resulted in a significant increase in rapid eye movement sleep, while slow wave sleep was unchanged. In contrast, adenosine perfusion into the laterodorsal tegmental nucleus produced an increase of both slow wave sleep and rapid eye movement sleep, the magnitude of which were proportional to the decrease in waking. Electroencephalographic power spectral analysis showed that adenosine perfusion into the basal forebrain increased the relative power in the delta frequency band, whereas higher frequency bands (theta, alpha, beta and gamma) showed a decrease. These data strongly support the hypothesis that adenosine might play a key role as an endogenous modulator of wakefulness and sleep. The decrease in wakefulness may be directly related to the inhibition of cholinergic neurons of the basal forebrain and the laterodorsal tegmentum. The increase in rapid eye movement sleep is a novel but robust effect whose origin, at present, is uncertain. The observation that local perfusion of adenosine into either the basal forebrain or the laterodorsal tegmental nucleus dramatically decreases wakefulness suggests that these areas might represent a major site of action of the xanthine stimulants (adenosine antagonists) found in coffee and tea.     

Cholinergic and non-cholinergic afferents of the caudolateral parabrachial nucleus: a role in the long-term enhancement of rapid eye movement sleep

A single microinjection of the cholinergic agonist carbachol into the feline caudolateral parabrachial nucleus produces an immediate increase in state-independent ipsilateral ponto-geniculooccipital waves, followed by a long-term rapid eye movement sleep enhancement lasting 7-10 days. Using retrogradely-transported fluorescent carbachol-conjugated nanospheres and choline acetyltransferase immunohistochemistry, afferent projections to this injection site for long-term rapid eye movement sleep enhancement were mapped and quantified. Six regions in the brain stem contained retrogradely-labelled cells: the raphe nuclei, locus coeruleus, laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, parabrachial nucleus, and the pontine reticular formation. The retrogradely-labelled (rhodamine+) cells in the pontine reticular formation and pedunculopontine tegmental nucleus contributed the predominant input to the parabrachial nucleus injection site (34.3 +/- 5.3% and 28.4 +/- 5.6%, respectively), compared to the laterodorsal tegmental nucleus (5.8 +/- 3.8%), parabrachial nucleus (13.5 +/- 3.1%), raphe nuclei (12.9 +/- 2.7%), and locus coeruleus (5.1 +/- 2.4%). By comparison with findings of afferent input to the induction site for short-latency rapid eye movement sleep in the anterodorsal pontine reticular formation, the parabrachial nucleus injection site is characterized by a similar proportion of afferents, except that the raphe nuclei were found to provide more than a two-fold greater input. Retrogradely-labelled neurons quantified in these nuclear regions consisted of 21.5% double-labelled (rhodamine+/choline acetyltransferase+) cholinergic and 78.5% noncholinergic (rhodamine+/choline acetyltransferase-) cells. The pedunculopontine tegmental nucleus contributed the predominant (51.7 +/- 8.2%) cholinergic input, compared to laterodorsal tegmental nucleus (20.7 +/- 10.2%), parabrachial nucleus (23.1 +/- 7.5%), and pontine reticular formation (4.4 +/- 2.1%). A comparative analysis of the total retrogradely-labelled cells within each nuclear region which were also double-labelled showed the highest proportion in the laterodorsal tegmental nucleus (76.2 +/- 7.5%) compared to pedunculopontine tegmental nucleus (39.4 +/- 3.6%), parabrachial nucleus (37.3 +/- 2.8%), and pontine reticular formation (3.2 +/- 2.1%). These data indicate that while pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus neurons exert a powerful cholinergic influence on the injection site for long-term rapid eye movement enhancement, a major component of the afferent circuitry is non-cholinergic. Since the non-cholinergic input includes contributions from the locus coeruleus and raphe nuclei, it is probable that the caudolateral parabrachial nucleus contains cholinergic and aminergic afferent systems that participate in the long-term enhancement of rapid eye movement sleep.     

Effects of excitation of sensory pathways on the membrane potential of cat masseter motoneurons before and during cholinergically induced motor atonia

Electrical stimulation of the nucleus pontis oralis during wakefulness enhances somatic reflex activity; identical stimuli during the motor atonia of active (rapid eye movement) sleep induces reflex suppression. This phenomenon, which is called reticular response-reversal, is based upon the generation of excitatory postsynaptic potential activity in motoneurons during wakefulness and inhibitory postsynaptic potential activity during the motor atonia of active sleep. In the present study, instead of utilizing artificial electrical stimulation to directly excite brainstem structures, we sought to examine the effects on motoneurons of activation of sensory pathways by exogenously applied stimuli (auditory) and by stimulation of a peripheral (sciatic) nerve. Accordingly, we examined the synaptic response of masseter motoneurons prior to and during cholinergically induced motor atonia in a pharmacological model of active sleep-specific motor atonia, the alpha-chloralose-anesthetized cat, to two different types of afferent input, one of which has been previously demonstrated to elicit excitatory motor responses during wakefulness. Following the pontine injection of carbachol, auditory stimuli (95 dB clicks) elicited a hyperpolarizing potential in masseter motoneurons. Similar responses were obtained upon stimulation of the sciatic nerve. Responses of this nature were never seen prior to the injection of carbachol. Thus, stimulation of two different afferent pathways (auditory and somatosensory) that produce excitatory motor responses during wakefulness instead, during motor atonia, results in the inhibition of masseter motoneurons. The switching of the net result of the synaptic response from one of potential motor excitation to primarily inhibition in response to the activation of sensory pathways was comparable to the phenomenon of reticular response-reversal. This is the first report to examine the synaptic mechanisms whereby exogenously or peripherally applied stimuli that elicit motor excitation during wakefulness instead elicit inhibitory motor responses during the motor atonia of active sleep. Thus, not only are motoneurons tonically inhibited during active sleep, but the selective elicitation of inhibitory motor responses indicates that this inhibition can be phasically increased in response to sensory stimuli, possibly in order to maintain the state of active sleep. The data provided the foundation for the hypothesis that, during naturally occurring active sleep, there is a change in the control of motor systems so that motor suppression occurs in response to stimuli that would otherwise, if present during other behavioral states, result in the facilitation of motor activity.     

Nitric oxide synthase immunoreactivity in rat pontine medullary neurons

Nitric oxide synthase immunoreactivity was detected in neurons and fibers of the rat pontine medulla. In the medulla, nitric oxide synthase-positive neurons and processes were observed in the gracile nucleus, spinal trigeminal nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, nucleus ambiguus, medial longitudinal fasciculus, reticular nuclei and lateral to the pyramidal tract. In the pons, intensely labeled neurons were observed in the pedunculopontine tegmental nucleus, paralemniscal nucleus, ventral tegmental nucleus, laterodorsal tegmental nucleus, and lateral and medial parabrachial nuclei. Labeled neurons and fibers were seen in the interpeduncular nuclei, dorsal and median raphe nuclei, central gray and dorsal central gray, and superior and inferior colliculi. Double-labeling techniques showed that a small population (< 5%) of nitric oxide synthase-positive neurons in the medulla also contained immunoreactivity to the aminergic neuron marker tyrosine hydroxylase. The majority of nitric oxide synthase-immunoreactive neurons in the dorsal and median raphe nuclei were 5-hydroxytryptamine-positive, whereas very few 5-hydroxytryptamine-positive cells in the caudal raphe nuclei were nitric oxide synthase-positive. Virtually all nitric oxide synthase-positive neurons in the pedunculopontine and laterodorsal tegmental nuclei were also choline acetyltransferase-positive, whereas nitric oxide synthase immunoreactivity was either low or not detected in choline acetyltransferase-positive neurons in the medulla. The results indicate a rostrocaudal gradient in the intensity of nitric oxide synthase immunoreactivity, i.e. it is highest in neurons of the tegmentum nuclei and neurons in the medulla are less intensely labeled. The majority of cholinergic and serotonergic neurons in the pons are nitric oxide synthase-positive, whereas the immunoreactivity was either too low to be detected or absent in the large majority of serotonergic, aminergic and cholinergic neurons in the medulla.     

Opioids activate G proteins in REM sleep-related brain stem nuclei of rat

Mu opioid receptors within the pontine reticular formation contribute to opioid-induced rapid eye movement (REM) sleep inhibition. Mu receptors are coupled to guanine nucleotide binding (G) proteins and this study tested the hypothesis that the micro opioid agonist [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) would activate G proteins in rat brain stem nuclei known to regulate REM sleep. In vitro autoradiography of DAMGO-stimulated [35S]GTPgammaS binding showed that, compared with basal [35S]GTPgammaS binding, DAMGO significantly increased G protein activation in the nucleus pontis oralis (56.2%), nucleus pontis caudalis (57.3%), laterodorsal tegmental nucleus (75.8%), pedunculopontine tegmental nucleus (72.4%), nucleus locus coeruleus (77.2%) and dorsal raphe nucleus (73.4%). DAMGO stimulation of [35S]GTPgammaS binding in nuclei regulating REM sleep suggests that opioid-induced REM sleep inhibition involves activation of G proteins.     

Behavioral state control through differential serotonergic inhibition in the mesopontine cholinergic nuclei: a simultaneous unit recording and microdialysis study

Cholinergic neurons of the mesopontine nuclei are strongly implicated in behavioral state regulation. One population of neurons in the cholinergic zone of the laterodorsal tegmentum and the pedunculopontine nuclei, referred to as rapid eye movement (REM)-on neurons, shows preferential discharge activity during REM sleep, and extensive data indicate a key role in production of this state. Another neuronal group present in the same cholinergic zone of the laterodorsal tegmentum and the pedunculopontine nuclei, referred to as Wake/REM-on neurons, shows preferential discharge activity during both wakefulness and REM sleep and is implicated in the production of electroencephalographic activation in both of these states. To test the hypothesis of differential serotonergic inhibition as an explanation of the different state-related discharge activity, we developed a novel methodology that enabled, in freely behaving animals, simultaneous unit recording and local perfusion of neuropharmacological agents using a microdialysis probe adjacent to the recording electrodes. Discharge activity of REM-on neurons was almost completely suppressed by local microdialysis perfusion of the selective 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), although this agonist had minimal or no effect on the Wake/REM-on neurons. We conclude that selective serotonergic inhibition is a basis of differential state regulation in the mesopontine cholinergic nuclei, and that the novel methodology combining neurophysiological and neuropharmacological information from the freely behaving animal shows great promise for further insight into the neural basis of behavioral control.     

In vitro release of acyclovir from semisolid dosage forms: effect of cyclodextrin and polyethylene glycol

Presynaptic depolarization of trigemino-thalamic (TGT) terminals may contribute to modulation of ascending oro-facial somatosensory information during active (or rapid eye movement) sleep. The relative excitability of TGT terminals was inferred from changes in the current required to maintain an antidromic firing probability of 50% (EC50) during quiet wakefulness as compared to active sleep. Depolarization or hyperpolarization of TGT terminals was defined as a decrease or increase, respectively, in the EC50. Overall, the EC50 of 8 TGT terminals was reduced by a mean 8.8+/-3.6 microA during active sleep relative to quiet wakefulness. This result suggests that depolarization of TGT terminals, which may act to suppress the transfer of sensory information from the trigeminal nucleus to the thalamus, occurs during active sleep.     

Response of locus coeruleus neurons to footshock stimulation is mediated by neurons in the rostral ventral medulla

While it is well documented that locus coeruleus neurons are potently activated by foot-pinch or sciatic nerve stimulation, little is known about the circuit producing this sensory response. Previous work in our laboratory has identified the medullary nucleus paragigantocellularis as a major excitatory afferent to the locus coeruleus. Here, we use local microinjections into the paragigantocellularis to test whether this nucleus is a link in the pathway mediating the activation of locus coeruleus neurons by subcutaneous footpad stimulation, or footshock, in anesthetized rats. Lidocaine HCl microinjected into the paragigantocellularis reversibly attenuated footshock-evoked activation of 50 out of 56 locus coeruleus cells, with responses in 20 cells completely blocked. Microinjections of GABA into the paragigantocellularis reduced the footshock-evoked responses of 17 out of 27 locus coeruleus cells (seven complete blocks); microinjections of the GABAB agonist baclofen had no effect (0 out of 11 cells blocked). Microinjections of a synaptic decoupling cocktail of manganese and cadmium also attenuated locus coeruleus activation in eight out of nine cells with two complete blocks. With each agent, the most effective injection placement for complete blockade of responses was the ventromedial paragigantocellularis; injections bordering this region attenuated responses, while those outside of the paragigantocellularis (dorsal medullary reticular formation, nucleus tractus solitarius, or facial nucleus), or vehicle injections, were ineffective. These results are consistent with previous findings that pharmacologic blockade of paragigantocellularis-evoked locus coeruleus activity also blocks footshock-evoked responses of locus coeruleus neurons [Ennis and Aston-Jones (1988) J. Neurosci. 8, 3644-3657], and support the view that this somatosensory response, and perhaps other sensory-evoked responses of locus coeruleus neurons, involve the nucleus paragigantocellularis.     

Influence of sleep states on laryngeal and abdominal muscle response to upper airway occlusion in lambs

This study was aimed at describing abdominal and laryngeal muscle responses to upper airway occlusion (UAO) in early life and the effect of sleep states on these responses. Twelve nonsedated, 9-26-d-old lambs were studied. We simultaneously recorded 1) airflow (pneumotachograph + face mask); 2) sleep states (electrocorticogram and electrooculogram); 3) abdominal muscle (external obliquus) electromyogram (EMG); and 4) glottic constrictor (thyroarytenoid) and dilator (posterior cricoarytenoid and cricothyroid) muscle EMGs. The pneumotachograph was repeatedly occluded for 15-30 s in wakefulness and natural sleep. We analyzed 90 occlusions during wakefulness (11 lambs), 28 during non-rapid eye movement (nREM) sleep (six lambs), and 23 during rapid eye movement (REM) sleep (five lambs). A phasic expiratory external obliquus EMG was present during baseline and progressively increased throughout UAO in wakefulness and nREM sleep, but not in REM sleep. Phasic thyroarytenoid EMG progressively increased during inspiratory efforts throughout UAO in wakefulness and nREM sleep, paralleling the increase in glottic dilator (posterior cricoarytenoid and cricothyroid) EMG. In contrast, glottic muscle response to UAO in REM sleep was severely blunted or disorganized by frequent swallowing movements. We conclude that UAO triggers complex and coordinated laryngeal and abdominal muscle responses during wakefulness and nREM sleep in lambs; these responses are largely absent, however, in REM sleep. These unique results, together with the defective arousal response in REM sleep, suggest that vulnerability to airway occlusion could be increased during REM sleep in early life. Possible implications for understanding severe postnatal apneas are discussed.     

Adenosine: a mediator of the sleep-inducing effects of prolonged wakefulness

Both subjective and electroencephalographic arousal diminish as a function of the duration of prior wakefulness. Data reported here suggest that the major criteria for a neural sleep factor mediating the somnogenic effects of prolonged wakefulness are satisfied by adenosine, a neuromodulator whose extracellular concentration increases with brain metabolism and which, in vitro, inhibits basal forebrain cholinergic neurons. In vivo microdialysis measurements in freely behaving cats showed that adenosine extracellular concentrations in the basal forebrain cholinergic region increased during spontaneous wakefulness as contrasted with slow wave sleep; exhibited progressive increases during sustained, prolonged wakefulness; and declined slowly during recovery sleep. Furthermore, the sleep-wakefulness profile occurring after prolonged wakefulness was mimicked by increased extracellular adenosine induced by microdialysis perfusion of an adenosine transport inhibitor in the cholinergic basal forebrain but not by perfusion in a control noncholinergic region.     

The influence of DNA glycosylases on spontaneous mutation

In the present study we examined the effects of phasic activation of the nucleus locus coeruleus (LC) on transmission of somatosensory information to the rat cerebral cortex. The rationale for this investigation was based on earlier findings that local microiontophoretic application of the putative LC transmitter, norepinephrine (NE), had facilitating actions on cortical neuronal responses to excitatory and inhibitory synaptic stimuli and more recent microdialysis experiments that have demonstrated increases in cortical levels of NE following phasic or tonic activation of LC. Glass micropipets were used to record the extracellular activity of single neurons in the somatosensory cortex of halothane-anesthetized rats. Somatosensory afferent pathways were activated by threshold level mechanical stimulation of the glabrous skin on the contralateral forepaw. Poststimulus time histograms were used to quantitate cortical neuronal responses before and at various time intervals after preconditioning burst activation of the ipsilateral LC. Excitatory and postexcitatory inhibitory responses to forepaw stimulation were enhanced when preceded by phasic activation of LC at conditioning intervals of 200-500 ms. These effects were anatomically specific in that they were only observed upon stimulation of brainstem sites close to (>150 micron) or within LC and were pharmacologically specific in that they were not consistently observed in animals where the LC-NE system had been disrupted by 6-OHDA pretreatment. Overall, these data suggest that following phasic activation of the LC efferent system, the efficacy of signal transmission through sensory networks in mammalian brain is enhanced.     

Progressive cortical synchronization of ponto-geniculo-occipital potentials during rapid eye movement sleep

Phasic events, termed ponto-geniculo-occipital potentials, appear in the brainstem, thalamus and cerebral cortex during rapid eye movement sleep. In the cat, the species of choice for ponto-geniculo-occipital studies, these field potentials are usually recorded from the lateral geniculate thalamic nucleus and visual cortex. However, the fact that brainstem cholinergic neurons play a crucial role in the transfer of ponto-geniculo-occipital potentials to the thalamus, coupled with the evidence that mesopontine tegmental neurons project to virtually all thalamic nuclei, together explain why ponto-geniculo-occipital potentials are recorded over widespread territories, beyond the visual thalamocortical system. Here we demonstrate, by means of multi-site unit and field potential recordings from sensory, motor and association cortical areas in behaving cats, that: (i) ponto-geniculo-occipital potentials appear synchronously over the neocortex; and (ii) that their cortical synchronization develops progressively from the period preceding rapid eye movement sleep by 30-90 s (pre-rapid eye movement), to reach the highest degree of intracortical coherence during later epochs of rapid eye movement sleep. We propose that the widespread coherence of cortical ponto-geniculo-occipital potentials underlies the synchronization of fast oscillations (30-40 Hz) during rapid eye movement sleep over many, functionally distinct cortical territories implicated in dreaming, as brainstem-induced ponto-geniculo-occipital-like potentials are consistently followed by such fast oscillations.     

Effect of strychnine on rat locus coeruleus neurones during sleep and wakefulness

The noradrenergic neurones of the locus coeruleus (LC) discharge tonically during wakefulness, decrease their activity during slow wave sleep and are virtually quiescent during paradoxical sleep. We recently demonstrated an inhibitory glycinergic input to the locus coeruleus and proposed that this could be responsible for inhibition of the LC during paradoxical sleep. To test this proposal, we developed a method combining polygraphic recordings, iontophoresis and single-unit extracellular recordings in the unanaesthetized head-restrained rat. Iontophoretically applied strychnine, a specific glycine antagonist, induced strong excitation of LC neurones during paradoxical sleep, but also during slow wave sleep and wakefulness. These results suggest that glycine tonically inhibits noradrenergic LC neurones throughout the entire sleep-waking cycle and not only during paradoxical sleep.     

Subpallidal outputs to the nucleus accumbens and the ventral tegmental area: anatomical and electrophysiological studies

The goal of this study was to investigate the functional organization of the subpallidal-->accumbens direct and indirect feedback loops by both anatomical and electrophysiological methods. The results of the dextran-conjugated rhodamine injections into the subpallidal area has shown three distinct projections: (1) a substantial pathway from the subpallidal area to the ventral tegmental area, (2) a more diffuse rostral projection from the subpallidal area to the core area of the nucleus accumbens, and (3) a sparse pathway projecting rostrodorsally from the subpallidal area toward the thalamic regions. Electrical or chemical stimulation of the subpallidal region, which was studied by the axonal tracer, evoked inhibitory responses in the majority (60 and 80%, respectively) of the accumbens and ventral tegmental area neurons in a standard extracellular recording study. Less than 1/3 of the accumbens or ventral tegmental area cells showed an increase in the mean firing rate. The majority (77.5%) of all responded neurons had a latency of less than 10 ms. Furthermore, injection of glutamate into the subpallidal area not only altered the firing pattern of the accumbens neurons, but also attenuated their excitatory responses elicited by the electrical stimulation of the ventral subiculum. Our results indicate that the subpallidal area plays a predominantly inhibitory role in the ventral tegmental area-accumbens-subpallidal circuitry, presumably by its GABAergic projections, and may also modulate subicular input into the nucleus accumbens.     

Voltage-clamp analysis and computer simulation of a novel cesium-resistant A-current in guinea pig laterodorsal tegmental neurons

Increased firing of cholinergic neurons of the laterodorsal tegmental nucleus (LDT) plays a critical role in generating the behavioral states of arousal and rapid eye movement sleep. The majority of these neurons exhibit a prominent transient potassium current (IA) that shapes firing but the properties of which have not been examined in detail. Although IA has been reported to be blocked by intracellular cesium, the IA in LDT neurons appeared resistant to intracellular cesium. The present study compared the properties of this cesium-resistant current to those typically ascribed to IA. Whole cell recordings were obtained from LDT neurons (n = 67) in brain slices with potassium- or cesium-containing pipette solutions. A transient current was observed in cells dialyzed with each solution (KGluc-85%; CsGluc-79%). However, in cesium-dialyzed neurons, the transient current was inward at test potentials negative to about -35 mV. Extracellular 4-aminopyridine (4-AP; 2-5 mM) blocked both inward and outward current, suggesting the inward current was reversed IA rather than an unmasked transient calcium current as previously suggested. This conclusion was supported by increasing [K]o from 5 to 15 mM, which shifted the reversal potential positively for both inward and outward current (+17.89 +/- 0.41 mV; mean +/- SE). Moreover, recovery from inactivation was rapid (tau = 15.5 +/- 4 ms; n = 4), as reported for IA, and both inward and outward transient current persisted in calcium-free solution [0 calcium/4 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid; n = 4] and during cadmium-blockade of calcium currents (n = 3). Finally, the transient current was blocked by intracellular 4-AP indicating that adequate dialysis occurred during the recordings. Thus the Cs-resistant current is a subthreshold IA. We also estimated the voltage-dependence of activation (V1/2 = -45.8 +/- 2 mV, k = 5.21 +/- 0.62 mV, n = 6) and inactivation (V1/2 = -59. 0 +/- 2.38 mV, k = -5.4 +/- 0.49 mV, n = 3) of this current. Computer simulations using a morphologically accurate model cell indicated that except for the extreme case of only distal A-channels and a high intracellular resistivity, our parameter estimates were good approximations. In conclusion, guinea pig LDT neurons express subthreshold A-channels that are resistant to intracellular cesium ions. This suggests that these channels differ fundamentally in their ion permeation mechanism from those previously studied. It remains to be determined if Cs+ resistance is common among brain A-channels or if this property is conferred by known A-channel subunits.     

Injection of corticotropin-releasing hormone into the locus coeruleus or foot shock increases neuronal Fos expression

Previous research suggests that corticotropin-releasing hormone can act in the locus coeruleus to increase the firing of locus coeruleus neurons and elicit physiological responses resembling those associated with stress. The present study used immunocytochemical detection of Fos as a measure of neuronal activation to identify brain areas that were activated by bilateral injections of corticotropin-releasing hormone into the locus coeruleus of rats. Injection of corticotropin-releasing hormone into the locus coeruleus increased the expression of Fos in the locus coeruleus as compared with injection of vehicle into the locus coeruleus or injection of corticotropin-releasing hormone into neighbouring pontine sites. The pattern of Fos expression throughout the brain after injections of corticotropin-releasing hormone into the locus coeruleus was generally consistent with the anatomical organization of efferent projections arising from the locus coeruleus; increased Fos expression was observed in many brain areas including the ventral lateral septum, septohypothalamic nucleus, bed nucleus of the stria terminalis, the central amygdaloid nucleus, the dorsomedial nuclei of the hypothalamus, and the thalamic paraventricular and rhomboid nuclei. Foot shock also increased Fos expression in the locus coeruleus and the other brain regions that expressed Fos after corticotropin-releasing hormone injections into the locus coeruleus. A few brain regions, most notably the hypothalamic paraventricular nucleus, expressed Fos in response to foot shock but not corticotropin-releasing hormone. These results indicate that local injection of corticotropin-releasing hormone into the locus coeruleus stimulates the activity of the locus coeruleus neurons. However, the pattern of Fos expression throughout the brain evoked by injection of corticotropin-releasing hormone into the locus coeruleus does not fully replicate the effects of foot shock.     

Contributions of the pedunculopontine region to normal and altered REM sleep

The pedunculopontine (PPN) region of the upper brainstem is recognized as a critical modulator of activated behavioral states such as wakefulness and rapid eye movement (REM) sleep. The expression of REM sleep-related physiology (e.g. thalamocortical arousal, ponto-geniculate-occipital (PGO) waves, and atonia) depends upon a subpopulation of PPN neurons that release acetylcholine (ACh) to act upon muscarinic receptors (mAChRs). Serotonin's potent hyperpolarization of cholinergic PPN neurons is central to present working models of REM sleep control. A growing body of experimental evidence and clinical experience suggests that the responsiveness of the PPN region, and thereby modulation of REM sleep, involves closely adjacent glutamatergic neurons and alternate afferent neurotransmitters. Although many of these afferents are yet to be defined, dopamine-sensitive GABAergic pathways exiting the main output nuclei of the basal ganglia and adjacent forebrain nuclei appear to be the most conspicuous and the most likely to be clinically relevant. These GABAergic pathways are ideally sited to modulate the physiologic hallmarks of REM sleep differentially (e.g. atonia versus cortical activation), because each originates from a functionally unique forebrain circuit and terminates in a unique pattern upon brain stem neurons with unique membrane characteristics. Evidence is reviewed that changes in the quality, timing, and quantity of REM sleep that characterize narcolepsy, REM sleep behavior disorder, and neurodegenerative and affective disorders (depression and schizophrenia) reflect 1) changes in responsiveness of cells in the PPN region governed by these afferents; 2) increase or decrease in PPN cell number; or 3) mAChRs mediating increased responsiveness to ACh derived from the PPN. Auditory evoked potentials and acoustic startle responses provide means independent from recording sleep to assess pathophysiologies affecting the PPN and its connections and thereby complement investigations of their role in affecting daytime functions (e.g. arousal and attention).     

Selection, optimization, and compensation as strategies of life management: correlations with subjective indicators of successful aging

To understand better how the brainstem may influence thalamocortical activity, we have examined the projection patterns of different brainstem nuclei to the thalamic reticular nucleus. Iontophoretic injections of biotinylated dextran were made into various nuclei of the brainstem (superior colliculus, periaqueductal grey matter, parabrachial nucleus, pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus, substantia nigra, ventral tegmental area, and locus coeruleus) of Sprague-Dawley rats by using stereotaxic coordinates. Our results show that afferents from each brainstem nucleus make distinct zones within the reticular nucleus. For example, the superior colliculus projects largely to the dorsal parts of the reticular nucleus, whereas the pedunculopontine nucleus projects to the ventral parts of the reticular nucleus. The substantia nigra, on the other hand, projects to the ventrolateral edge of the reticular nucleus. We also examined the distribution of these brainstem afferents within the dorsal thalamus and compared these distributions with those seen in the reticular nucleus. We found three different patterns. First, a given brainstem nucleus projects to a particular dorsal thalamic nucleus as well as to the corresponding, functionally associated, reticular sector (e.g., from the substantia nigra). Second, a given brainstem nucleus projects to a particular dorsal thalamic nucleus but not to the corresponding reticular sector (e.g., from the superior colliculus). Finally, a given brainstem nucleus projects to a given reticular sector but not to the corresponding dorsal thalamic nucleus (e.g., from the midbrain reticular nucleus). In general, our results indicate that various brainstem nuclei project to particular territories of the thalamic reticular nucleus. Through these reticular projections, brainstem nuclei may influence distinct thalamocortical pathways in addition to those that are influenced by their direct projection to the dorsal thalamus.