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Serotonin and noradrenaline modulate respiratory pattern disturbance evoked by glutamate injection into pedunculopontine tegmentum of anesthetized rats
Abstract
We hypothesized that 2 important neurotransmitters related to behavioral state control, serotonin and noradrenaline, could also be modulators of pedunculopontine tegmental nucleus (PPT)-induced respiratory dysrhythmia.
We examined the impact of serotonin and noradrenaline at respiratory control sites in the PPT functionally identified by immediate apnea of 2.5- to 10-second duration, followed by increased variability of breath time (CVT(T)) (P < .04) after locally injecting glutamate in anesthetized rats.
Basic sleep and respiratory neurobiology laboratory.
Sixteen adult, male Sprague-Dawley rats.
Glutamate-induced respiratory responses, including increases of total apnea duration and CVT(T), were not different between groups of rats in which we further tested monoaminergic modulatory effects (for CVT(T) P = .98, and for total apnea duration, P = .80). Serotonin or noradrenaline injected at the same sites as glutamate had equal impact on CVT(T) (P = .34) and on mean total apnea duration (P = .80), but pretreatment of PPT sites with serotonin blocked (remained equal to preinjection; P = .11), whereas pretreatment with noradrenaline potentiated (P = .04) the increment of respiratory-timing variability induced by glutamate. The serotonergic-blocking effect on glutamate-induced respiratory dysrhythmia was specific to the PPT: the respiratory responses induced by glutamate injection outside the PPT were not modulated by serotonin (for CVT(T), P = .46, and for mean apnea duration, P = .99).
The opposed impact of serotonin and noradrenaline on PPT-induced respiratory dysrhythmia, in contrast to their convergent regulatory role in behavioral state control, suggests a functionally distinct role for the PPT in respiratory-pattern control independent of rapid eye movement sleep control.
... Continuous electrical stimulation within the PPT evoked respiratory depression in anesthetized cats (Lydic and Baghdoyan, 1993), whereas we demonstrated that carbachol injection into the PPT increased respiratory dysrhythmia during sleep in conscious rats (Carley and Radulovacki, 1999;Radulovacki et al., 2004). We further showed that microinjection of glutamate into the PPT of anesthetized rats initiated respiratory disturbances characterized by irregular alternations between tachypnea and bradypnea/apnea (Saponjic et al., 2003;2005;2006). Stimulation of the PPT can also evoke cardiovascular reactions, characterized by increased blood pressure (BP) (Padley et al., 2007). ...
... Our laboratory has demonstrated that local injection of glutamate in anesthetized rats can evoke respiratory dysrhythmia, electroencephalogram (EEG) activation, hippocampal thetarhythm, or increased phasic events such as p-waves (Saponjic et al., 2003;2005;2006). Further, these studies suggested that such phenomena are at least partially differentiable, according to stimulation site, suggesting a functional topography. ...
... Injection volumes were directly measured using a dissecting microscope (Wild Heerbrugg, model M5) with a calibrated reticule to observe the movement of the fluid meniscus within the pipette barrel. The dose of the glutamate was chosen according to our previous work (Saponjic et al., 2003;2005;2006), which showed the effectiveness of this volume and concentration to evoke a prominent respiratory reaction from the PPT. Further, work by Nicholson (1985) suggests that within the first 35 s the effective diffusion radius is limited to approximately 150 µm. ...
Functionally distinct areas were mapped within the pedunculopontine tegmentum (PPT) of 42 ketamine/xylazine anesthetized rats using local stimulation by glutamate microinjection (10 mM, 5-12 nl). Functional responses were classified as: (1) apnea; (2) tachypnea; (3) hypertension (HTN); (4) sinus tachycardia; (5) genioglossus electromyogram activation or (6) pontine-waves (p-waves) activation.We found that short latency apneas were predominantly elicited by stimulation in the lateral portion of the PPT, in close proximity to cholinergic neurons. Tachypneic responses were elicited from ventral regions of the PPT and HTN predominated in the ventral portion of the antero-medial PPT. We observed sinus tachycardia after stimulation of the most ventral part of the medial PPT at the boundary with nucleus reticularis pontis oralis, whereas p-waves were registered predominantly following stimulation in the dorso-caudal portion of the PPT. Genioglossus EMG activation was evoked from the medial PPT. Our results support the existence of the functionally distinct areas within the PPT affecting respiration, cardiovascular function, EEG and genioglossus EMG.
... These breathing disturbances were observed to occur independently of any other REM sleep signs [35], suggesting that respiratory modulation by the PPT does not depend on brain phasic events. Nonetheless, the PPT respiratorymodulating region appears to be differentially regulated by two important REM sleep-related neurotransmitters (noradrenaline and serotonin) [36]. Collectively, these findings support a functionally distinct, but potentially state-dependent, respiratory-modulating region within the PPT. ...
... Significant attention has been focused on the PPT region as a potential mediator of REM-related breathing disorder because (1) PPT has distinct neuronal populations that are specifically activated during REM sleep and other populations that are selectively active during REM sleep and wakefulness [16]; and (2) PPT cholinergic neurons exhibit direct projections to the region of the respiratory pattern generator within the medulla [39]. Our previous work supports the possibility of direct respiratory pattern modulation by PPT neurons [14,35,36] and suggests that the relevant neuronal subpopulation may be differentially modulated by serotonin and noradrenaline-two key neuromodulators which are released maximally during wakefulness and minimally during REM sleep [40]. Together, these studies suggest that withdrawal of serotonergic damping of a critical neuronal pool within PPT during REM sleep may predispose to respiratory disturbance [36]. ...
... Our previous work supports the possibility of direct respiratory pattern modulation by PPT neurons [14,35,36] and suggests that the relevant neuronal subpopulation may be differentially modulated by serotonin and noradrenaline-two key neuromodulators which are released maximally during wakefulness and minimally during REM sleep [40]. Together, these studies suggest that withdrawal of serotonergic damping of a critical neuronal pool within PPT during REM sleep may predispose to respiratory disturbance [36]. The present Considerable progress has been made in identifying the connectivity and synaptic regulation of PPT cholinergic cells [16]. ...
The pedunculopontine tegmental nucleus (PPT) is postulated to have important functions relevant to the regulation of rapid eye movement (REM) sleep and arousal, and various motor control systems including respiration. We have recently shown that pharmacologic activation of a neuronal subpopulation within the PPT, induced by micropipette injection of glutamate in nanoliter volumes, can produce respiratory rhythm disturbances and changes in genioglossus muscle activity in anesthetized rats. The aim of this study was to determine whether the respiratory pattern disturbance and increased genioglossus muscle tone induced by glutamate injection within the PPT are mediated by activation of N-methyl-D-aspartate (NMDA) receptors within the PPT. Experiments were performed in eight adult male spontaneously breathing Sprague-Dawley rats anesthetized using nembutal. Respiratory movements were monitored by piezoelectric strain gauge. Three-barrel glass pipettes were used to pressure inject glutamate (as a probe for respiratory modulating sites), ketamine (an NMDA channel blocker), and oil-red dye (to aid in histological verification of the injection sites) within the PPT. Electroencephalograms were recorded from the sensorimotor cortex, the hippocampus, and the pons, contralateral to the injection site. Electromyograms (EMGs) were recorded from the genioglossus muscle. The typical response to glutamate injection within the PPT respiratory-modulating region was immediate apnea followed by tachypnea and increased genioglossal tonic activity. The noncompetitive NMDA receptor channel-antagonist ketamine, injected at the same site and in the same volume as glutamate (5 nl), blocked respiratory dysrhythmia and genioglossal EMG responses to subsequent glutamate injections. For the first time, the present results suggest that respiratory rhythm and upper airway muscle tone are controlled by the activation of pedunculopontine tegmental nucleus NMDA receptors.
... The pedunculopontine tegmental nucleus (PPT), as the main brain source of thalamo-cortical cholinergic innervation, is postulated to have important functions relevant to the regulation of rapid eye movement (REM) sleep (Lu et al., 2006), arousal (Datta and MacLean, 2007), and various motor control systems (Takakusaki et al., 2004), including respiratory pattern control and respiratory pattern variability and apneas during REM sleep (Lydic and Baghdoyan, 1993;Saponjic et al., 2003Saponjic et al., , 2005aSaponjic et al., ,b, 2006. In addition, the direct projections from the PPT reach both the basal forebrain and the thalamus (Losier and Semba, 1993), and therefore the PPT represents a control relay nucleus for the integrated contributions of these two cholinergic systems to the regulation of cortical activation (Dringenberg and Olmstead, 2003). ...
... In all the experimental groups, during 20 min of anesthesia, we recorded EEG and respiratory movements using the piezo electric strain gauge (Infant-Ped Sleepmate Technologies, Midlothain, VA, USA). This system comprised a 1-cm 2 crystal attached to an elastic band that was fixed around the animal at a substernal level (Saponjic et al., 2003(Saponjic et al., , 2005a(Saponjic et al., ,b, 2006. This crystal provided quantitative measurements of respiratory timing and relative measurements of respiratory volume, allowing us to track changes over time. ...
Objectives
We hypothesized that the impact of distinct anesthetic regimens could be differently expressed during anesthesia and on post-anesthesia sleep in the neurodegenerative diseases. Therefore, we followed the impact of ketamine/diazepam and pentobarbital anesthesia in a rat model of the severe Parkinson’s disease cholinergic neuropathology on the electroencephalographic (EEG) microstructure and respiratory pattern during anesthesia, and on the post-anesthesia sleep.
Methods
We performed the experiments on adult, male, spontaneously breathing Wistar rats chronically instrumented for sleep recording. The bilateral pedunculopontine tegmental nucleus (PPT) lesion was done by ibotenic acid microinfusion. Following postoperative recovery, we recorded sleep for 6 h, induced anesthesia 24 h later using ketamine/diazepam or pentobarbital, and repeated sleep recordings sessions 48 h and 6 days later. During 20 min of each anesthesia we recorded both the EEG and respiratory movements. For sleep and EEG analysis, Fourier analysis was applied on 6 h recordings, and each 10 s epoch was differentiated as a state of wakefulness (Wake), non-rapid eye movement (NREM) or rapid eye movement (REM). Additionally, the group probability density distributions of all EEG frequency band relative amplitudes were calculated for each state, with particular attention during anesthesia. For respiratory pattern analysis we used Monotone Signal Segments Analysis. The PPT lesion was identified through nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry.
Results and Conclusions
Our data show that the ketamine/diazepam anesthetic regimen in the PPT lesioned rats induces more alterations in the EEG microstructure and respiratory pattern than does the pentobarbital anesthesia. In addition, the equal time required to establish an anesthetized state, and the long-term effect on post-anesthesia sleep in the PPT lesioned vs. control rats suggest this anesthetic regimen as potentially more beneficial both for anesthesia induction and for post-anesthesia sleep in the surgical procedures of the elderly, and Parkinson’s, and Alzheimer’s patients.
... This study also provides functional evidence that respiratory disturbances induced by glutamate injection into the PPT of anesthetized rats can occur in the absence of other electrical, motor, or behavioral responses characteristic of a rapid eye movement (REM) sleep-like state. In accordance , our recent study has shown that two monoamines , with convergent roles in behavioral state control, exert opposite effects on PPT-evoked respiratory dis- turbances [42]. Collectively, these observations suggest a functionally distinct PPT subregion for respiratory pattern control independent of REM sleep control [42] . ...
... In accordance , our recent study has shown that two monoamines , with convergent roles in behavioral state control, exert opposite effects on PPT-evoked respiratory dis- turbances [42]. Collectively, these observations suggest a functionally distinct PPT subregion for respiratory pattern control independent of REM sleep control [42] . Stimulating the appropriate PPT region by local glutamate injection under anesthesia produced immediate apnea followed by increased respiratory timing variability lasting at least 2 min. ...
The pedunculopontine tegmental nucleus (PPT) has been shown to have important functions relevant to the regulation of behavioral states and various motor control systems, including breathing control. Our previous work has shown that the activation of neurons within the PPT, a structure that is typically active during rapid eye movement (REM) sleep, can produce respiratory disturbances in freely moving and anesthetized rats. The aim of this study was to test the hypothesis that respiratory modulation by the PPT in anesthetized rats can be evoked in the absence of other signs of an REM-sleep-like state. We characterized electroencephalogram (EEG) and electromyogram (EMG) changes during respiratory disturbances induced by glutamatergic stimulation of the PPT in spontaneously breathing, adult male Sprague-Dawley rats anesthetized with a ketamine/xylazine combination or with nembutal. Respiratory movements were monitored by a piezoelectric strain gauge. Two-barrel glass pipettes were used to pressure inject glutamate, to probe for respiratory effective sites within the PPT, and to inject oil red dye at the end of the experiments for histological verification of the injection sites. The EEGs were recorded from the sensorimotor cortex, hippocampus, and from the pons contralateral from the injection site. The EMGs were recorded from the genioglossus muscle. The initial response to glutamate injection into the respiratory modulating region of the PPT was always a respiratory pattern disturbance. Subsequent activation of EMG and EEG often occurred in ketamine/xylazine-anesthetized rats, but REM-sleep-like patterns were not observed. Respiratory pattern and EMG power changes in nembutal-anesthetized rats were similar, but EEG activation was never observed. Thus, we conclude that respiratory suppression produced by the local activation of PPT neurons may not necessarily be accompanied by an REM-sleep-like cortical state in this anesthetized model.
... We recently demonstrated anatomically distinct sites within the PPT that separately modulate P-wave activity and respiratory pattern Physiology & Behavior 90 (2007) 1 -10 [10,11]. Respiratory disturbances following glutamate injection into this "respiratory modulating region" of the PPT [10,12] are strongly and oppositely modulated by 5-HT and NA injection [14]. Thus, monoamine release by LC and DR may have similar functions in REM sleep and P-wave gating, but opposing roles on respiratory modulation by the PPT [14,15]. ...
... Respiratory disturbances following glutamate injection into this "respiratory modulating region" of the PPT [10,12] are strongly and oppositely modulated by 5-HT and NA injection [14]. Thus, monoamine release by LC and DR may have similar functions in REM sleep and P-wave gating, but opposing roles on respiratory modulation by the PPT [14,15]. ...
Monoamines are important regulators of behavioral state and respiratory pattern, and the impact of monoaminergic control during sleep is of particular interest for the stability of breathing regulation. The aim of this study was to test the effects of systemically induced chemical lesions to noradrenergic and serotonergic efferent systems, on the expression of sleep-wake states, pontine wave activity, and sleep-related respiratory pattern and its variability. In chronically instrumented male adult Sprague-Dawley rats we lesioned noradrenergic terminal axonal branches by a single intraperitoneal dose of DSP-4 (N-(2-chloroethyl)-N-ethyl-2-brombenzilamine; 50 mg/kg, i.p.), and serotonergic axonal terminals by two intraperitoneal doses, 24 h apart, of PCA (p-chloroamphetamine; 6 mg/kg, i.p.). In each animal, we recorded sleep, pontine waves (P-waves) and breathing at baseline, following sham injection, and every week for 5 weeks following injection of either systemic neurotoxin. Distinct responses were observed to the two lesions. DSP-4 lesions were associated with a trend toward increased NREM sleep (p < 0.06), decreased wakefulness (p < 0.05) and increased respiratory tidal volume during NREM (p = 0.0002) and REM (p = 0.0001) sleep with respect to baseline. None of these effects, however, were observed during the first 14 days after injection. No significant changes were observed in the frequency of apneas or sighs, nor in the coupling between these two, at any time after DSP-4 injection. Conversely, selective serotonergic lesion by PCA produced no change in the baseline respiratory frequency or tidal volume during sleep or wakefulness, nor was the expression of Wake, NREM or REM sleep affected. Instead, PCA injection resulted in a sustained increase in the frequency and duration of post-sigh apneas (PS) during NREM sleep (p = 0.002). This reflected increased coupling between sighs and apneas, because neither the frequency nor the amplitude of spontaneous sighs was altered by PCA.
... We have to note here, that the PPT has important functions relevant to the regulation of REM sleep [12,13], arousal [14], and various motor control systems [15], including breathing control [16,17]. It is also postulated that PPT is the high relay nucleus for overall REM sleep phenomenon control, and that each REM sleep event, executed by distinct cell groups within the brainstem, may be triggered and modulated by the activation of the PPT [18]. ...
... Moreover, based on a number of the animal studies [50][51][52], the PPT has become the main target for deep brain stimulation for the treatment of movement and postural disorders in humans [53]. PPT as the main brain source of thalamocortical cholinergic innervation has an important functions relevant to the regulation of REM sleep [54,55], arousal [55,56], and various motor control systems [53,57], including breathing control [58][59][60]. In addition, in relation to motor and cognitive function control, particularly in relation to Parkinson's disease, the PPT is postulated as an important interface between the basal ganglia and cerebellum [61]. ...
Rhythmic oscillations of neuronal populations, generated by different mechanisms, are present at several levels of the central nervous system and serve many important physiological or reflect pathological functions. Understanding the role of brain oscillations as possible biomarkers of brain function and plasticity is still a challenge, and despite extensive research, their role is still not well established.
We recently demonstrated that the hallmarks of earlier aging onset during impaired thalamo-cortical cholinergic innervation (in a rat model of Parkinson’s disease cholinopathy) were consistently expressed, from 3 and one half to 5 and one half months of age, through increased electroencephalographic (EEG) sigma activity amplitude during rapid eye movement (REM) sleep, as a unique aging induced REM sleep phenomenon. In addition, there was altered motor cortical drive during non-rapid-eye-movement (NREM) and REM sleep.
In order to explain this new aging-induced REM sleep phenomenon, we analyzed possible differences between control REM sleep spindle activity and REM sleep spindle activity at the onset of REM sleep “enriched“ with sigma activity (at 4 and one half months of age), following bilateral pedunculopontine tegmental nucleus (PPT) cholinergic neuronal loss in the rat. We analzyed differences in spindle density, duration, and frequency.
We demonstrated in young adult Wistar rats with the severely impaired PPT cholinergic innervation the alterations in sleep spindle dynamics and pattern during REM sleep in the motor cortex as the earliest biomarkers for the onset of their altered aging processes.
... Because direct projections from the PPT reach both the basal forebrain and thalamus, this nucleus is an ideal candidate to study the integrated contributions of these two systems to regulating activation of the neocortex [18]. PPT is postulated to have important functions relevant to the regulation of REM sleep [19,20], arousal [21][22][23][24][25][26][27][28][29], and various motor control systems [30][31][32], including breathing control [33][34][35][36][37][38]. In addition, the PPT has a central role in the REM sleep phenomenon control [39][40][41], and each individual REM-sleep-sign generating nucleus receives afferent inputs from PPT [28]. ...
The medical profession has been generally very slow to acknowledge the importance of sleep medicine and sleep research. Disorders of sleep are related to anxiety, many mental and neurodegenerative diseases, cardiovascular and respiratory disorders, and obesity.
Our knowledge of the neural substrates of sleep/wake states and sleep-related behavior disorders regulation in health and the diseases, over more than 50 years of sleep research, is based on the experiments in animal models, pharmacotherapy, and the neuropathological studies in humans. But, we still need further work in fundamental multidisciplinary and clinical research between sleep and neurodegenerative disease investigators to understand normal and abnormal sleep, and to provide new insights into preventive or disease-altering approaches for therapy.
Our aim is to give an overview of our recent results related to the importance of thalamo-cortical cholinergic brain system in the disorders of sleep and motor control during sleep, with particular relevance to Parkinson’s disease.
... The cholinergic system in the mammalian brain consists of two distinct aggregations of cholinergic neurons: the basal forebrain complex, which involves the NB, substantia innominata, and the horizontal limb of the diagonal band, and the brainstem complex with the PPT, as the major aggregation of cholinergic neurons [21][22][23]. While the NB has a direct role in cortical activation and sleep homeostasis [24], the PPT, as the main brain source of thalamo-cortical cholinergic innervation, is postulated to have important functions relevant to the regulation of REM sleep [25,26], arousal [26,27], and various motor control systems [28,29], including breathing control [30][31][32][33]. Anatomical studies support the central role of the PPT in controlling the REM sleep phenomenon [21,22], and show that direct projections from PPT reach both the basal forebrain and the thalamus [34], and therefore the PPT represents the control relay nucleus for the integrated contributions of these two cholinergic systems to the regulation of cortical activation [24,35]. ...
... The cholinergic system in the mammalian brain consists of two distinct aggregations of cholinergic neurons, the basal forebrain complex, which involves the nucleus basalis (NB), the substantia innominata, and the horizontal limb of the diagonal band, and the brainstem complex, with the pedunculopontine tegmental nucleus (PPT) as the major aggregation of cholinergic neurons (Semba and Fibiger, 1989;Semba, 1993;Rye, 1997). Whereas the NB has a direct role in cortical activation and sleep homeostasis (Dringenberg and Olmstead, 2003), the PPT is important in the regulation of arousal (Datta and MacLean, 2007), various motor control systems (Takakusaki et al., 2004), breathing control (Lydic and Baghdoyan, 1993;Saponjic et al., 2003Saponjic et al., , 2005aSaponjic et al., ,b, 2006, and generation and maintenance of REM sleep (Semba, 1993;Rye, 1997;Lu et al., 2006;Datta and MacLean, 2007). ...
Alzheimer's disease (AD) involves selective loss of basal forebrain cholinergic neurons, particularly in the nucleus basalis (NB). Similarly, Parkinson's disease (PD) might involve the selective loss of pedunculopontine tegmental nucleus (PPT) cholinergic neurons. Therefore, lesions of these functionally distinct cholinergic centers in rats might serve as models of AD and PD cholinergic neuropathologies. Our previous articles described dissimilar sleep/wake-state disorders in rat models of AD and PD cholinergic neuropathologies. This study further examines astroglial and microglial responses as underlying pathologies in these distinct sleep disorders. Unilateral lesions of the NB or the PPT were induced with rats under ketamine/diazepam anesthesia (50 mg/kg i.p.) by using stereotaxically guided microinfusion of the excitotoxin ibotenic acid (IBO). Twenty-one days after the lesion, loss of cholinergic neurons was quantified by nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, and the astroglial and microglial responses were quantified by glia fibrillary acidic protein/OX42 immunohistochemistry. This study demonstrates, for the first time, the anatomofunctionally related astroglial response following unilateral excitotoxic PPT cholinergic neuronal lesion. Whereas IBO NB and PPT lesions similarly enhanced local astroglial and microglial responses, astrogliosis in the PPT was followed by a remote astrogliosis within the ipslilateral NB. Conversely, there was no microglial response within the NB after PPT lesions. Our results reveal the rostrorostral PPT-NB astrogliosis after denervation of cholinergic neurons in the PPT. This hierarchically and anatomofunctionally guided PPT-NB astrogliosis emerged following cholinergic neuronal loss greater than 17% throughout the overall rostrocaudal PPT dimension. © 2014 Wiley Periodicals, Inc.
... The pedunculopontine tegmental nucleus (PPT), being the main brain source of the thalamo-cortical cholinergic innervation, is postulated to have important functions relevant to the regulation of REM [9], arousal [10], and various motor control systems [11], including breathing control [12][13][14][15]. PPT is also postulated as the high relay nucleus for overall REM sleep phenomenon control, and each REM sleep event, executed by distinct cell groups within the brainstem, may be triggered and modulated by the activation of the PPT [10]. ...
... Since the cholinergic NB projections to the cortex are not diffuse, but modality and region-specific – distinct populations of cholinergic neurons are capable of modulating different cortical regions [15]. The PPT is postulated to have important functions relevant to the regulation of REM161718, arousal [19], and various motor control systems [20], and breathing control2122232425 , and the direct projections from PPT reach both the basal forebrain and thalamus [26]. Therefore, the PPT presents the control relay nucleus for the integrated contributions of these two cholinergic systems to regulation of cortical activation [27,28]. ...
... Lesions of the nucleus basalis magnocellularis (NB) in vivo have been widely used as an animal model for understanding the role of cholinergic neurotransmission in the central nervous system functions, and the disorders caused by cortical cholinergic neuronal degeneration such as Alzheimer's disease (Arendash et al., 1987;Anezaki et al., 1992;Monzon-Mayor et al., 2000;Alvarez 2004, 2010;Swarowsky et al., 2008;Topchiy et al., 2010). Local stereotaxically guided nanovolume microinfusion/ microinjection technique in vivo enable us to pharmacologically stimulate, lesion, trace, and generally to investigate in animals the functional topography of certain brain nucleus with a resolution of ≤ 10 µm, and at a level of 300 µm of effective radius within the brain tissue Alvarez 2004, 2010;McKay et al., 2005;Saponjic et al., 2003Saponjic et al., , 2005aSaponjic et al., , 2005bLu et al., 2006;Swarowsky et al., 2008;Topchiy et al., 2010). The advantage of local stimulation by glutamate or DL homocysteic acid microinjection, or local lesion by microinfusion of the excitotoxins (glutamate, ibotenic acid (IBO), IgG saporin), over electrical stimulation/lesion of the same neuronal population, is that they reduce the likelihood of activation/lesion of fibers of passage (Monnier et al., 2003). ...
Our study investigates the impact of different volume sham control and excitotoxin microinfusions in vivo on local reactive astroglial response within rat nucleus basalis (NB). We followed the effects of unilateral 200, 100, and 50 nL of sham-control (phosphate buffer PBS) versus ibotenic acid (IBO) microinfusions, mechanical NB lesion (10 µL Hamylton syringe needle positioned into NB for 5 min), or physiological control (intact brain), on the local reactive astroglial response within the NB site, by immunoreactivity against glial fibrillary acidic protein (GFAP). NB lesions were identified by NADPHdiaphorase histochemistry. Local astrocytes responses within NB were suppressed by both high volume microinfusions, PBS and IBO (200 and 100 nL) versus mechanical lesion. Our study has proved, for the first time, the volume of microinfusion as critical for any selective pharmacological stimulation or lesion in vivo, and suggest the microinfusion volume less than 50nL as protective for physiological astroglial reactivity.
... The pedunculopontine tegmental nucleus (PPT) is postulated to have important functions relevant to the regulation of rapid eye movement sleep (REM) (Lu et al., 2006; McCarley and Hobson, 1975), arousal (Bringmann, 1995Bringmann, , 1997 Datta, 2002; Datta and MacLean, 2007; Datta and Siwek, 1997; Datta et al., 2001; Kleiner and Bringmann, 1996; Rye, 1997; Vertes, 1984), and various motor control systems (Inglis et al., 1994; Saper and Loewy, 1982; Takakusaki et al., 2004), including breathing control (Lydic and Baghdoyan, 1993; Radulovacki et al., 2004; Saponjic et al., 2003 Saponjic et al., , 2005a Saponjic et al., , 2005b Saponjic et al., , 2006). It is also postulated that PPT is the high relay nucleus for overall REM sleep phenomenon control, and that each REM sleep event, executed by distinct cell groups within the brainstem, may be triggered and modulated by the activation of the PPT (Datta, 1995Datta, ,1997 Garcia-Rill, 1991). ...
... Inhibitory interaction between these regions are important in regulating their activity, and the opposite, reciprocal discharge patterns of the monoaminergic and cholinergic neurons within pons were proposed to underline the cyclic appearance of REM sleep (McCarley and). DR and LC are the main sources of monoamines in the brain, and their extensive efferents implicate these structures in a variety of behavioral functions, particularly in sleep regulation (Lydic et al. 1987), motor (Fenik and Veasey 2003) and respiratory control (Carley and Radulovacki 2003; Saponjic et al. 2005). It was demonstrated that the systemic selective lesion of the noradrenergic or serotonergic axon terminals did not affect the sleep/wake distribution compared with controls (Saponjic et al. 2007a). ...
This study was aimed to explore the sleep/wake states related cortico-pontine theta carrier frequency phase shift following a systemically induced chemical axotomy of the monoaminergic afferents within a brain of the freely moving rats. Our experiments were performed in 14 adult, male Sprague Dawley rats, chronically implanted for sleep recording. We recorded sleep during baseline condition, following sham injection (saline i.p. 1 ml/kg), and every week for 5 weeks following injection of the systemic neurotoxins (DSP-4 or PCA; 1 ml/kg, i.p.) for chemical axotomy of the locus coeruleus (LC) and dorsal raphe (DR) axon terminals. After sleep/wake states identification, FFT analysis was performed on 5 s epochs. Theta carrier frequency phase shift (∆Φ) was calculated for each epoch by averaging theta Fourier component phase shifts, and the ∆Φ values were plotted for each rat in control condition and 28 days following the monoaminergic lesions, as a time for permanently established DR or LC chemical axotomy. Calculated group averages have shown that ∆Φ increased between pons and cortex significantly in all sleep/wake states (Wake, NREM and REM) following the monoaminergic lesions, with respect to controls. Monoaminergic lesions established the pontine leading role in the brain theta oscillations during all sleep/wake states.
... In the ventral r1 field, the dorsal raphe is medial, the dorsal tegmental nucleus is in deep ventral r1 near the ventricle, while the pedunculopontine tegmental nucleus is more lateral and superficially localized (Martin, 2003;Schambra et al., 1992). Based on GABAergic identity and localization, we predict that PITX2-positive cells contribute to the pedunculopontine tegmental nucleus which is thought to be involved in local inhibition controlling locomotion, REM, alertness, and respiratory patterns (Datta et al., 2001;Kozak et al., 2005;Saponjic et al., 2005;Tsang et al., 2010). A modulatory role for PITX2-positive GABAergic neurons in respiration, alertness, or other important autonomic functions could help explain why Nestin-Cre conditional Pitx2 mutants fail to survive beyond the immediate postnatal period (Sclafani et al., 2006). ...
Hindbrain rhombomere 1 (r1) is located caudal to the isthmus, a critical organizer region, and rostral to rhombomere 2 in the developing mouse brain. Dorsal r1 gives rise to the cerebellum, locus coeruleus, and several brainstem nuclei, whereas cells from ventral r1 contribute to the trochlear and trigeminal nuclei as well as serotonergic and GABAergic neurons of the dorsal raphe. Recent studies have identified several molecular events controlling dorsal r1 development. In contrast, very little is known about ventral r1 gene expression and the genetic mechanisms regulating its formation. Neurons with distinct neurotransmitter phenotypes have been identified in ventral r1 including GABAergic, serotonergic, and cholinergic neurons. Here we show that PITX2 marks a distinct population of GABAergic neurons in mouse embryonic ventral r1. This population appears to retain its GABAergic identity even in the absence of PITX2. We provide a comprehensive map of markers that places these PITX2-positive GABAergic neurons in a region of r1 that intersects and is potentially in communication with the dorsal raphe.
... These norepinephrinergic (NE) neurons modulate firing activity of brain stem respiratory neurons (Biancardi et al. 2008). Breathing activity is affected by local injection of NE to the pedunculopontine tegmental nucleus (Saponjic et al. 2005). NE has excitatory effects on neurons in the pre-Bötzinger complex (PBC) (Doi and Ramirez 2010), a population of cells critical for respiratory control. ...
Catecholaminergic neurons in the locus coeruleus (LC) play a role in the ventilatory response to hypercapnia. Here, we show evidence for the involvement of transient receptor potential (TRP) channels. We found that the input resistance was reduced during an exposure to 8% CO(2) in ~35% LC neurons in mouse brain slices, accompanied by depolarization and higher firing activity. The neuronal responses suggest the opening of Na(+) or nonselective cationic channels instead of the closure of K(+) channels. As a major group of cationic channels, the TRP channels are expressed in the brain, some of which are activated by acidic pH. We therefore screened all representative TRP channels using the quantitative real-time PCR analysis. High levels of mRNA expression of TRPC5, TRPM2, and TRPM7 were found in the LC tissue. Of them, the TRPC5 transcript was the most abundant. The TRPC5 channel was activated by extracellular acidification when expressed in human embryonic kidney (HEK) cells. The TRPC5 currents started to be activated at pH 7.4 with pKa 6.9. The TRPC5 currents were also activated by isohydric hypercapnic and intracellular acidosis in a Ca(2+)-dependent manner. Consistently, the LC neurons were stimulated by both extra- and intracellular acidosis. The stimulatory effect of hypercapnia on LC neurons was eliminated by selective TRPC inhibitor SKF-96365 with and without the blockade of synaptic transmission. Single-cell PCR analysis indicated that TRPC5 mRNAs existed in the LC neurons. Thus these results strongly suggest that the TRP channels are likely to play a role in the CO(2) chemosensitivity of LC neurons, especially TRPC5.
... DR and LC are the main sources of monoamines in the brain. Their extensive efferents implicate these structures in a variety of behavioral functions, particularly in sleep regulation (20,22), motor and respiratory control (23)(24)(25). ...
The experiments were performed in 14 adult, male Sprague Dawley rats chronically instrumented for sleep recording and recorded during baseline condition, following sham injection (saline i.p. 1 ml/kg), and every week for 5 weeks following injection of the systemic neurotoxins (DSP-4 or PCA; 1 ml/kg, i.p.) for chemical axotomy of the locus coeruleus (LC) and dorsal raphe (DR) axon terminals. In our former study we demonstrated that the systemically induced lesion of the noradrenergic or serotonergic axon terminals did not affect the sleep-wake distribution from control condition. In this study, by using spectral analysis and phase shift spectra of the cortical and pontine EEG we analyzed cortico-pontine theta oscillation synchronization phase shift on 6-hour recordings in control condition and 28 days following the monoaminergic lesions, as a time for permanently established DR or LC chemical axotomy. Our results demonstrated for the first time that chronically decreased brain monoamines in freely moving rats changed cortico-pontine theta synchronization phase shift. Pons became a leading theta oscillator. We assume that deficit of monoamines induced predominance of the NREM/REM transitions, characterized with phasic theta oscillations (the increased density of clustered P waves which intrinsic frequency corresponds to theta frequency oscillations), and may produced preceding phasic theta versus tonic theta oscillation drive.
The technologies of genomics and proteomics are powerful tools for discovering novel gene and protein expression responses to disease. Considerable evidence indicates that a genetic basis exists to the causes of sleep-disordered breathing, in particular its most common form of obstructive sleep apnea (OSA), which is characterized by periods of intermittent hypoxia and disrupted sleep. However, the genetic contribution to the pathogenesis of OSA has largely been determined using traditional genetic approaches of family, twin, and linkage studies in clinical populations and quantitative trait loci and targeted gene procedures in animal models of OSA. In contrast to the pathogenesis of OSA, the consequences or sequelae of OSA are highly amenable to genomic and proteomic approaches. Animal studies have assessed changes in gene and protein expression in multiple organ systems in response to intermittent hypoxia and sleep deprivation and uncovered novel gene activation paradigms. The first tentative steps have been made toward applying proteomic analyses of blood and urine from patients with OSA as a potential screening tool for diagnosis in the clinical setting. It is anticipated that genomic and proteomic technologies will become increasingly used in the area of OSA with the unprecedented access to tissue in procedures such as bariatric surgery. OSA represents a severe insult to the oxygenation of tissues and the homeostasis of sleep, and genomic and proteomic approaches hold promise for defining previously unexplored mechanisms and pathways that lead to downstream pathologies, including hypertension, insulin resistance, and neurocognitive dysfunction.
Serotonergic suppression of cholinergic neuronal activity implicated in the regulation of rapid eye movement sleep and its associated phenomenon, pontogeniculooccipital waves, has long been postulated, but no direct proof has been available. In this study, intracellular and whole-cell patch-clamp recording techniques were combined with enzyme histochemistry to examine the intrinsic electrophysiological properties and response to serotonin (5-HT) of identified cholinergic rat laterodorsal tegmental nucleus neurons in vitro. Sixty-five percent of the recorded neurons demonstrated a prominent low-threshold burst, and of these, 83% were cholinergic. In current-clamp recordings 64% of the bursting cholinergic neurons tested responded to the application of 5-HT with a membrane hyperpolarization and decrease in input resistance. This effect was mimicked by application of the selective 5-HT type 1 receptor agonist carboxamidotryptamine maleate. Whole-cell patch-clamp recordings revealed that the hyperpolarizing response was mediated by an inwardly rectifying K+ current. Application of 5-HT decreased excitability and markedly modulated the discharge pattern of cholinergic bursting neurons: during a 5-HT-induced hyperpolarization these neurons exhibited no rebound burst after hyperpolarizing current input and a burst in response to depolarizing current input. In the absence of 5-HT, the relatively depolarized cholinergic bursting neurons responded to an identical hyperpolarizing current input with a burst and did not produce a burst after depolarizing current input. These data provide a cellular and molecular basis for the hypothesis that 5-HT modulates rapid eye movement sleep phenomenology by altering the firing pattern of bursting cholinergic neurons.
As originally named for the ostensibly contradictory appearance of rapid eye movements and low voltage fast cortical activity during behavioral sleep, paradoxical sleep or rapid eye movement sleep, represents a distinct third state, in addition to waking and slow wave sleep, in mammals and birds. It is an internally generated state of intense tonic and phasic central activation that is contemporaneous with the inhibition of sensory input and motor output. In early studies, it was established that the state of paradoxical sleep was generated within the brainstem, and particularly within the pons. Pharmacological studies indicated an important role for acetylcholine as a neurotransmitter in the generation of this state. Local injections of cholinergic agonists into the pontine tegmentum triggered a state of paradoxical sleep marked by phasic ponto-geniculo-occipital spikes in association with cortical activation and neck muscle atonia. Following the immunohistochemical identification of choline acetyl transferase-containing neurons and their localization to the dorsolateral ponto-mesencephalic tegmentum, neurotoxic lesions of this major cholinergic cell group could be performed to assess its importance in paradoxical sleep. Destruction of the majority of the cholinergic cells, which are concentrated within the laterodorsal tegmental and pedunculopontine tegmental nuclei but extend also into the locus coeruleus and parabrachial nuclei in the cat, resulted in a loss or diminishment of the state of paradoxical sleep, ponto-geniculo-occipital spiking and neck muscle atonia. These deficits were correlated with the loss of choline acetyltransferase-immunoreactive neurons in the region, so as to corroborate results of pharmacological studies and single unit recording studies indicating an active role of these cholinergic cells in the generation of paradoxical sleep and its components. These cells provide a cholinergic innervation to the entire brainstem reticular formation that may be critical in the generation of the state which involves recruitment of massive populations of reticular neurons. Major ascending projections into the thalamus, including the lateral geniculate, may provide the means by which phasic (including ponto-geniculo-occipital spikes) and tonic activation is communicated in part to the cerebral cortex. Descending projections through the caudal dorsolateral pontine tegmentum and into the medial medullary reticular formation may be involved in the initiation of sensorimotor inhibition. Although it appears that the pontomesencephalic cholinergic neurons play an important, active role in the generation of paradoxical sleep, this role may be conditional upon the simultaneous inactivity of noradrenaline and serotonin neurons, evidence for which derives from both pharmacological and recording studies.(ABSTRACT TRUNCATED AT 400 WORDS)
Spontaneous discharge of norepinephrine-containing locus coeruleus (NE-LC) neurons was examined during the sleep-walking cycle (S-WC) in behaving rats. Single unit and multiple unit extracellular recordings yielded a consistent set of characteristic discharge properties. (1) Tonic discharge co-varied with stages of the S-WC, being highest during waking, lower during slow wave sleep, and virtually absent during paradoxical sleep. (2) Discharge anticipated S-WC stages as well as phasic cortical activity, such as spindles, during slow wave sleep. (3) Discharge decreased within active waking during grooming and sweet water consumption. (4) Bursts of impulses accompanied spontaneous or sensory-evoked interruptions of sleep, grooming, consumption, or other such ongoing behavior. (5) These characteristic discharge properties were topographically homogeneous for recordings throughout the NE-LC. (6) Phasic robust activity was synchronized markedly among neurons in multiple unit populations. (7) Field potentials occurred spontaneously in the NE-LC and were synchronized with bursts of unit activity from the same electrodes. (8) Field potentials became dissociated from unit activity during paradoxical sleep, exhibiting their highest rates in the virtual absence of impulses. These results are generally consistent with previous proposals that the NE-LC system is involved in regulating cortical and behavioral arousal. On the basis of the present data and those described in the following report (Aston-Jones, G., and F. E. Bloom (1981) J. Neurosci.1: 887-900), we conclude that these neurons may mediate a specific function within the general arousal framework. In brief, the NE-LC system may globally bias the responsiveness of target neurons and thereby influence overall behavioral orientation.
Previous evidence has suggested that neurons in the medial medullary reticular formation play a critical role in the modulation of forebrain and spinal cord activity that occurs during the sleep-waking cycle and particularly in association with the state of paradoxical sleep. The importance of these neurons, including cholinergic, serotonergic and GABAergic cells [Holmes C. J. et al. (1994) Neuroscience 62, 1155–1178] for sleep-wake states was investigated after their destruction with the neurotoxin quisqualic acid injected into the medullary gigantocellular and magnocellular tegmental fields in cats. To assess the effects of the neuronal loss, polygraphic recording and behavioural observations were performed in baseline and for three weeks after the lesion, and the changes in these measures were correlated with the volume of destruction of medullary regions and the numbers of chemically identified cells within those regions.
The parabrachial complex, also known as the pneumotaxic center or pontine respiratory group, has long been recognized as an important participant in respiratory control. One line of evidence supporting this idea is the demonstration of changes in breathing pattern following injection of neuroactive substances into or near the parabrachial complex. However, it is not yet known exactly which cell groups and projections mediate those responses. In order to address this issue, we explored the topographic organization of respiratory responses to chemical stimulation of the parabrachial complex of the rat and examined the descending projections of the most sensitive sites. Injection of glutamate (5-100 pmol) at specific sites in or near the parabrachial nucleus produced three distinct site-specific response patterns. First, hyperpnea followed glutamate injection into far rostral and midcaudal areas of the Kölliker-Fuse nucleus and most of the lateral parabrachial nucleus, including the external lateral, central lateral, dorsal lateral, and superior lateral subnuclei. Threshold hyperpneic effects were manifested as single, deepened breaths of premature onset. Suprathreshold doses of glutamate at these locations produced tachypnea. Neurons in these sites projected to the ventral respiratory group in the ventrolateral medulla. Second, the most intense inspiratory facilitatory responses were seen at mid to rostral levels of the Kölliker-Fuse nucleus, near the ventrolateral tip of the superior cerebellar peduncle. Even at threshold doses of glutamate, exhalation was incomplete, resulting in a breathing pattern that resembled apneusis (an inspiratory cramp). This site contained an especially dense cluster of neurons that projected either to the ventrolateral medulla or to the dorsal respiratory group in the nucleus of the solitary tract, but not to both areas. The third type of response, decreases in respiratory rate, occurred following glutamate injection at the most lateral and ventral boundaries of the Kölliker-Fuse nucleus. The most sensitive apneic sites were not found in the parabrachial nucleus but along the dorsal and medial edge of the principal sensory trigeminal nucleus and extending ventrally between the sensory and motor trigeminal nuclei. Scattered neurons in these sites were retrogradely labeled from the ventral but not the dorsal respiratory group. These results indicate that there are anatomically and functionally distinct cell populations in and near the parabrachial complex that, when chemically stimulated, can produce specific and sometimes opposing effects on respiration. The predominant effect of lateral parabrachial stimulation is respiratory facilitation, while inhibitory effects are elicited by trigeminal injections of glutamate.
Inhibition of brainstem cholinergic neurons by noradrenergic neurons of the locus ceruleus has long been suggested as a key mechanism of behavioral state control. In particular, the commonly held view is that noradrenaline (NA) plays a permissive role in rapid eye movement (REM) sleep generation by disinhibiting brainstem cholinergic neurons. While this notion has been supported by numerous investigations, the inhibition of cholinergic neurons by NA has never been directly demonstrated. The purpose of this study was to investigate the effects of NA upon identified cholinergic neurons in the rat mesopontine tegmentum. Using whole-cell patch-clamp recordings in slices, 175 cells were studied during bath application of 50 microM NA. Cholinergic neurons were positively identified by intracellular labeling with biocytin and subsequent staining with NADPH-diaphorase, a reliable marker for brainstem cholinergic neurons (Vincent et al., 1983). Successful intracellular labeling was obtained in 96 cells. Ninety-two percent (36 of 39) of cholinergic neurons hyperpolarized in response to NA, while noncholinergic cells (n = 57) exhibited mixed responses. Application of NA in a low-Ca2+, high-Mg2+ solution elicited the same hyperpolarizing effect as in normal solution, which indicated that the effect of NA on cholinergic neurons was direct. The noradrenergic hyperpolarization was mimicked by the alpha 2-adrenoceptor agonist UK-14,304, and was blocked by the alpha 2-adrenoceptor antagonist idazoxan, which suggested an alpha 2-mediated response. Finally, voltage-clamp experiments revealed that NA activates the inwardly rectifying potassium current, IKG.(ABSTRACT TRUNCATED AT 250 WORDS)
1. The pedunculopontine tegmental (PPT) cholinergic nucleus and the locus coeruleus (LC) noradrenergic nucleus were electrically stimulated to investigate their effects on the recently described slow oscillation (approximately 0.3 Hz) of neocortical neurons. Intracellular recordings of slowly oscillating, regular-spiking and intrinsically bursting neurons from cortical association areas 5 and 7 (n = 140) were performed in anesthetized cats. 2. Pulse trains to the PPT nucleus produced the blockage of rhythmic (approximately 0.3 Hz) depolarizing-hyperpolarizing sequences in 79% of tested cortical neurons and transformed this slow cellular rhythm into tonic firing. The latency of the cortical cellular response to PPT stimulation was 1.2 +/- 0.5 (SE) s and its duration was 15.9 +/- 1.9 s. The PPT-elicited suppression of the slow cellular oscillation was accompanied by an activation of the electroencephalogram (EEG) having a similar time course. Fast Fourier transform analyses of EEG activities before and after PPT stimulation showed that the PPT-evoked changes consisted of decreased power of slow rhythms (0-8 Hz) and increased power of fast rhythms (24-33 Hz); these changes were statistically significant. 3. The blockage of the slow cellular oscillation was mainly achieved through the diminution or suppression of the long-lasting hyperpolarizations separating the rhythmic depolarizing envelopes. This effect was observed even when PPT pulse trains disrupted the oscillation without inducing overt depolarization and increased firing rate. The durations of the prolonged hyperpolarizations were measured during a 40-s window (20 s before and 20 s after the PPT pulse train) and were found to decrease from 1.5 +/- 0.2 to 0.7 +/- 0.1 s. The values of the product resulting from the duration (in seconds), the amplitude (in millivolts), and number of such hyperpolarizing events within 20-s periods were 51.5 +/- 5 and 5.1 +/- 1.9 before and after PPT stimulation, respectively. 4. The PPT effect was suppressed by systemic administration of a muscarinic antagonist, scopolamine, but not by mecamylamine, a nicotinic antagonist. 5. The PPT effect on cellular and EEG cortical slow oscillation survived, although its duration was reduced, in animals with kainate-induced lesions of thalamic nuclei projecting to areas 5 and 7 (n = 3) as well as in animals with similar excitotoxic lesions leading to extensive neuronal loss in nucleus basalis (n = 2). These data indicate that the PPT effect is transmitted to neocortex through either thalamic or basal forebrain relays.(ABSTRACT TRUNCATED AT 400 WORDS)
The present study examined the hypothesis that cholinergic neurons in the pedunculopontine tegmental nucleus (PPT) can cause the release of acetylcholine (ACh) in the pontine reticular formation and contribute to respiratory depression. In vivo microdialysis of the gigantocellular tegmental field (FTG) was performed in 10 adult male cats while respiration was being measured. In four intact, unanesthetized cats these measurements were obtained during states of quiet wakefulness and during the rapid eye movement (REM) sleeplike state caused by FTG microinjections of carbachol. The results demonstrate a simultaneous time course of enhanced ACh release in the FTG and respiratory rate depression. In six barbiturate-anesthetized cats similar measurements were obtained while PPT regions containing NADPH-positive neurons were electrically stimulated. PPT stimulation caused increased ACh release in the FTG and caused respiratory rate depression. Together, these findings are consistent with the hypothesis of a causal relationship between ACh release in the FTG and respiratory depression.
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.
Cholinergic neurons of the pontomesencephalic tegmentum play a critical role in paradoxical sleep, when, according to single unit recording of 'possibly' cholinergic neurons, they fire maximally. The profile of activity of the cholinergic neurons may be determined by noradrenergic locus coeruleus neurons that are active during waking and silent during paradoxical sleep. Indeed, a permissive role of the noradrenergic neurons in paradoxical sleep has been proposed based upon an inhibitory action of noradrenaline through alpha(2) adrenergic receptors on the cholinergic cells. Yet some 'possibly' cholinergic neurons are purportedly maximally active during waking and excited by noradrenaline through alpha(1) receptors. In the present study, we examined by fluorescent dual-immunostaining in the laterodorsal and pedunculopontine tegmental nuclei of the rat whether choline acetyltransferase-immunopositive neurons are stained for alpha(2A) or alpha(1A) adrenergic receptors. For comparison, we examined immunostaining for these receptors on tyrosine hydroxylase-immunopositive locus coeruleus neurons, which are known to bear alpha(2A) autoreceptors. Whereas virtually all the noradrenergic neurons were labeled for the alpha(2A) and none for the alpha(1A), approximately half the cholinergic neurons were labeled for the alpha(2A) and one third for the alpha(1A) adrenergic receptors in adjacent sections. These results suggest that different groups of cholinergic neurons bear alpha(2) versus alpha(1) adrenergic receptors and would accordingly have different sleep-wake state activities and roles. The alpha(2)-bearing group would be inhibited by noradrenaline during waking to become disinhibited and maximally active while promoting paradoxical sleep, whereas the alpha(1)-bearing group would be excited by noradrenaline during waking to become maximally active while promoting features of that state.
The last two decades have witnessed an exponential increase in knowledge regarding sleep-related breathing disorders (SRBD). Significant strides have been made in our understanding of these disorders with respect to epidemiology and risk factors, pathogenesis, clinical and behavioral consequences, and appropriate diagnostic and treatment strategies. Still, work to understand these factors in terms of the underlying cellular and molecular processes is in its infancy. As detailed in the subsequent chapters of this volume, fundamental tools and approaches of molecular biology and quantitative neuroscience are now being employed to study SRBDs. The next decade should mark important advances in this area, with Significant progress in therapies directed at the specific pathophysiology of these disorders.
An important challenge to the field today is to determine the neural bases for sleep-related changes in control of respiration. Animal models are essential in this endeavor and recent advances have been made in the studies of breathing disorders in marine mammals, preterm lambs, English bulldogs, and neonatal and mature rats. Our group has illustrated this strategy, by characterizing the respiratory instability and its neural mechanisms in the rat model of sleep apnea. In addition, our studies have indicated pharmacologic avenues for the treatment of central apneas in the rat, central/obstructive apneas in other animal species, and the potential management of human sleep apnea syndrome.
During the sleep cycle in cats, neurons localized to the posterolateral pole of the nucleus locus coeruleus and the nucleus
subcoeruleus undergo discharge rate changes that are the opposite of those of the pontine reticular giant cells. The inverse
rate ratios and activity curves of these two interconnected populations are compatible with reciprocal interaction as a physiological
basis of sleep cycle oscillation.
Increasingly strong evidence suggests that cholinergic neurons in the mesopontine tegmentum play important roles in the control of wakefulness and sleep. To understand better how the activity of these neurons is regulated, the potential afferent connections of the laterodorsal (LDT) and pedunculopontine tegmental nuclei (PPT) were investigated in the rat. This was accomplished by using retrograde and anterograde axonal transport methods and NADPH-diaphorase histochemistry. Immunohistochemistry was also used to identify the transmitter content of some of the retrogradely identified afferents.
The pedunculopontine tegmental nucleus (PPTg) contains a population of cholinergic neurons thought to be part of the ascending reticular activating system, and non-cholinergic neurons. In the previous study it was shown that various excitotoxins made effective lesions of cholinergic neurons in the PPTg but that quinolinate made smaller lesions in the non-cholinergic population, making it more selective than any other excitotoxin. The purpose of the present experiment was, first, to make lesions of cholinergic neurons throughout the length of the PPTg by infusing toxin at two different sites within it; and second, to examine simple motor activities in rats bearing either quinolinate or ibotenate lesions of the PPTg, and contrast these with the deficits seen after 6-hydroxydopamine (6-OHDA) induced lesions of mesostriatal dopamine (DA)-containing neurons. Post-mortem examination was carried out using choline acetyltransferase (ChAT) and tyrosine hydroxylase (TOH) immunohistochemistry, and routine Nissl staining. Both quinolinate and ibotenate destroyed approximately 75% of ChAT-positive neurons in the PPTg, but damage to non-cholinergic neurons (assessed by Nissl staining) was twice as great following ibotenate as quinolinate. 6-OHDA induced almost complete lesions of mesostriatal DA neurons, assessed by TOH immunohistochemistry. DA depleted rats showed deficits in drinking and spilled more food in the first 2 weeks after surgery, and were unable to reach or grasp food pellets in the staircase test. They also showed strong ipsilateral turning in response to amphetamine and contralateral turning to apomorphine. Quinolinate lesioned rats had no eating or drinking impairment in the home cage but showed a reaching (though not grasping) disability in the staircase test. They had a mild ipsilateral bias following amphetamine. Ibotenate lesioned rats, despite having larger lesions than the quinolinate, showed no deficits in eating or drinking in the home cage, or reaching or grasping disabilities in the staircase test. They did have a mild contralateral bias in response to amphetamine. This dissociation of the effects of quinolinate and ibotenate lesions of the PPTg is consistent with the suggestion that the PPTg has two functionally distinct components, and is attributed to the differential lesion of non-cholinergic neurons by the two excitotoxins.
The afferent connections of the pedunculopontine tegmental nucleus (PPT) and the adjacent midbrain extrapyramidal area (MEA) were examined by retrograde tracing with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Major afferents to the PPT originate in the periaqueductal gray, central tegmental field, lateral hypothalamic area, dorsal raphe nucleus, superior colliculus, and pontine and medullary reticular fields. Other putative inputs originate in the paraventricular and preoptic hypothalamic nuclei, the zona incerta, nucleus of the solitary tract, central superior raphe nucleus, substantia innominata, posterior hypothalamic area, and thalamic parafascicular nucleus. The major afferent to the medially adjacent MEA originates in the lateral habenula, while other putative afferents include the perifornical and lateral hypothalamic area, periaqueductal gray, superior colliculus, pontine reticular formation, and dorsal raphe nucleus. MEA inputs from basal ganglia nuclei include moderate projections from the substantia nigra pars reticulata, entopeduncular nucleus, and a small projection from the globus pallidus, but not the subthalamic nucleus. Dense anterograde labeling was observed in the substantia nigra pars compacta, entopeduncular nucleus, subthalamic nucleus, globus pallidus, and caudate-putamen only following WGA-HRP injections involving the MEA.
The ultrastructure of choline acetyltransferase (ChAT)-immunoreactive neurons in the laterodorsal tegmental nucleus (TLD) of the rat was investigated by immunohistochemical techniques. The immunoreactive neurons were medium to large in size, with a few elongated dendrites, contained well-developed cytoplasm, and a nucleus with deep infoldings. They received many nonimmunoreactive, mostly asymmetric synaptic inputs on their soma and dendrites. ChAT-immunoreactive, usually myelinated, axons were occasionally seen in TLD. Only one immunoreactive axon terminal was observed within TLD, and it made synaptic contact with a nonimmunoreactive neuronal perikaryon. The synaptic interactions between ChAT-immunoreactive neurons and tyrosine hydroxylase (TH)-immunoreactive fibers in the TLD were investigated with a double immunohistochemical staining method. ChAT-immunoreactivity detected with a beta-galactosidase method was light blue-green in the light microscope and formed dot-like electron dense particles at the electron microscopic level. TH-immunoreactivity, visualized with a nickel-enhanced immunoperoxidase method, was dark blue-black in the light microscope and diffusely opaque in the electron microscope. Therefore, the difference between these two kinds of immunoreactivity could be quite easily distinguished at both light and electron microscopic levels. In the light microscope, TH-positive fibers were often closely apposed to ChAT-immunoreactive cell bodies and dendrites in TLD. In the electron microscope, the cell soma and proximal dendrites of ChAT-immunoreactive neurons received synaptic contacts from TH-immunoreactive axon terminals. These results provide a morphological basis for catecholaminergic regulation of the cholinergic reticular system.
The cholinergic agonist carbachol was injected into the pontine Pb area where PGO bursting cells have been recorded. When microinjections were localized to the ventrolateral aspect of the caudal Pb nucleus near aggregates of ChAT immunolabeled cholinergic neurons, carbachol produced an immediate onset of state-independent PGO waves in the ipsilateral LGB. These state-independent PGO waves persisted for 3-4 days. After the first 24 hrs PGO wave activity increasingly became associated with REM sleep and with REM transitional SP sleep as both of these PGO-related states increased in amount to 3-4 times baseline levels. The increase in amount of PGO-related states peaked on days 2-4 following one carbachol injection and persisted for 10-12 days. These results suggest a two stage process: stage one, PGO enhancement, is the direct consequence of the membrane activation of cholinoceptive PGO burst neurons by carbachol; stage two, REM enhancement, is the consequence of metabolic activation of endogenous cholinergic neurons. This experimental preparation is a useful model for the study of the electrophysiology and functional significance of PGO wave and REM sleep generation.
Transection, lesion and unit recording studies have localized rapid eye movement (REM) sleep mechanisms to the pons. Recent work has emphasized the role of pontine cholinergic cells, especially those of the pedunculopontine tegmentum (PPT). The present study differentiated REM sleep deficits associated with lesions of the PPT from other pontine regions implicated in REM sleep generation, including those with predominantly cholinergic vs non-cholinergic cells. Twelve hour polygraphic recordings were obtained in 18 cats before and 1-2 weeks after bilateral electrolytic or radio frequency lesions of either: (1) PPT, which contains the dorsolateral pontine cholinergic cell column; (2) laterodorsal tegmental nucleus (LDT), which contains the dorsomedial pontine cholinergic cell column; (3) locus ceruleus (LC), which contains mostly noradrenergic cells; or (4) subceruleus (LC alpha, peri-LC alpha and the lateral tegmental field), which also contains predominantly noncholinergic cells. There were three main findings: (i) Only lesions of PPT and subceruleus significantly affected REM sleep time. These lesions produced comparable reductions in REM sleep time but influenced REM sleep components quite differently: (ii) PPT lesions, estimated to damage 90 +/- 4% of cholinergic cells, reduced the number of REM sleep entrances and phasic events, including ponto-geniculooccipital (PGO) spikes and rapid eye movements (REMs), but did not prevent complete atonia during REM sleep: (iii) Subceruleus lesions eliminated atonia during REM sleep. Mobility appeared to arouse the cat prematurely from REM sleep and may explain the brief duration of REM sleep epochs seen exclusively in this group. Despite the reduced amount of REM sleep, the total number of PGO spikes and REM sleep entrances increased over baseline values. Collectively, the results distinguish pontine loci regulating phasic events vs atonia. PPT lesions reduced phasic events, whereas subceruleus lesions created REM sleep without atonia. Severe REM sleep deficits after large pontine lesions, including PPT and subceruleus, might be explained by simultaneous production of both REM sleep syndromes. However, extensive loss of ACh neurons in the PPT does not disrupt REM sleep atonia.
For the last decade the functional organization pf cholinergic neurons has dominated studies of the basal forebrain. Cholinergic neurons in the brain, exclusive of motor neurons and interneurons, are found in two spatially separate groups (Armstrong et al., 1983, Mesulam et al., 1984). The rostral group, located in the basal forebrain, has received substantial attention because of its corticopedal projections (Mesulam et al., 1984) and its’ degeneration in Alzheimer’s disease (Coyle et al., 1983). The caudal group is found in the laterodorsal tegmental nucleus (LDT) and pedunculopontine nucleus (PPT) within the pontine tegmentum (Vincent et al., 1983; Mesulam et al., 1984; Satoh and Fibiger, 1986), and is the source of cholinergic innervation to the basal forebrain, thalamus and brainstem (Sofroniew et al., 1985; Hallenger et al., 1987; Maley et al., 1988; Rye et al., 1988; Jones, 1990).
Studies of the pedunculopontine (PPT) and laterodorsal tegmental (LDT) nuclei in the mesopontine tegmentum have emphasized the organization and projections of the cholinergic neurons. We report here that exhibiting glutamate immunoreactivity are present in both the LDT and PPT. These glutamatergic neurons are interspersed among the cholinergic neurons within both nuclei with no apparent segregation. These data raise the possibility that excitatory amino acids contribute to the functions of the LDT and PPT.
The pedunculopontine tegmental nucleus (PPTn) was originally defined on cytoarchitectonic grounds in humans. We have employed cytoarchitectonic, cytochemical, and connectional criteria to define a homologous cell group in the rat. A detailed cytoarchitectonic delineation of the mesopontine tegmentum, including the PPTn, was performed employing tissue stained for Nissl substance. Choline acetyltransferase (ChAT) immunostained tissue was then analyzed in order to investigate the relationship of cholinergic perikarya, dendritic arborizations, and axonal trajectories within this cytoarchitectonic scheme. To confirm some of our cytoarchitectonic delineations, the relationships between neuronal elements staining for ChAT and tyrosine hydroxylase were investigated on tissue stained immunohistochemically for the simultaneous demonstration of these two enzymes.
The PPTn consists of large, multipolar neurons, all of which stain immunohistochemically for ChAT. It is present within cross‐sections that also include the A‐6 through A‐9 catecholamine cell groups and is traversed by catecholaminergic axons within the dorsal tegmental bundle and central tegmental tract. The dendrites of PPTn neurons respect several nuclear boundaries and are oriented perpendicularly to several well‐defined fiber tracts. Cholinergic axons ascend from the mesopontine tegmentum through the dorsal tegmental bundle and a more lateral dorsal ascending pathway. A portion of the latter terminates within the lateral geniculate nucleus.
It has been widely believed that the PPTn is reciprocally connected with several extrapyramidal structures, including the globus pallidus and substantia nigra pars reticulata. Therefore, the relationships of pallidotegmental and nigrotegmental pathways to the PPTn were investigated employing the anterograde autoradiographic methodology. The reciprocity of tegmental connections with the substantia nigra and entopeduncular nucleus was investigated employing combined WGA‐HRP injections and ChAT immunohistochemistry.
The pallido‐ and nigrotegmental terminal fields did not coincide with the PPTn, but, rather, were located just medial and dorsomedial to it (the midbrain extrapyramidal area). The midbrain extrapyramidal area, but not the PPTn, was reciprocally connected with the substantia nigra and entope‐duncular nucleus. We discuss these results in light of other cytoarchitec‐tonic, cytochemical, connectional, and physiologic studies of the functional anatomy of the mesopontine tegmentum.
When a substance is pressure-injected from a micropipette into the extracellular space of the brain it may either form a cavity or it may infiltrate the extracellular space. In either case subsequent diffusion is governed by the volume fraction and tortuosity of the brain tissue as well as the diffusion coefficient of the substance itself. Appropriate equations, solutions and approximations to these problems are discussed. The results are relevant to the interpretation of studies on neuropharmacology and in situ electrochemistry.
Previous studies in our laboratory have shown that microinjection of acetylcholine and non-N-methyl-D-aspartate (NMDA) glutamate agonists into the pontine inhibitory area (PIA) induce muscle atonia. The present experiment was designed to identify the PIA afferents that could be responsible for these effects, by use of retrograde transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP), glutamate immunohistochemistry and NADPH-diaphorase staining techniques. Experiments were performed in both decerebrate and intact cats. Dense retrograde WGA-HRP labelling was found in neurons in the periaqueductal gray (PAG) and mesencephalic reticular formation (MRF) at the red nucleus (RN) level, ventral portion of paralemniscal tegmental field (vFTP), retrorubral nucleus (RRN), contralateral side of PIA (cPIA), pontis reticularis centralis caudalis (PoC), and most rostral portion of the nucleus parvicellularis (NPV) and nucleus praepositus hypoglossi (PH) at the level of the pontomedullary junction; moderate labelling was seen in pedunculopontine nucleus, pars compacta (PPNc), laterodorsal tegmental nucleus (LDT), superior colliculus (SC), MRF and PAG at the level caudal to RN, medial and superior vestibular nuclei, and principle sensory trigeminal nucleus (5P); and light labelling was seen in dorsal raphe (DR) and locus coeruleus complex (LCC). The projection neurons were predominantly ipsilateral to the injection site, except for both vFTP and RRN, which had more projection cells on the contralateral side. Double labelled WGA-HRP/NADPH-d neurons could be found in PPNc and LDT. Double labelled WGA-HRP/glutamatergic neurons could be seen at high densities in MRF, RRN, vFTP, and cPIA, moderate densities in SC, LDT, PPNc, PoC, and NPV, and low densities in PH, 5P, DR, LCC, and PAG.(ABSTRACT TRUNCATED AT 250 WORDS)
Extensive studies have ascribed a role to the brainstem cholinergic system in the generation of rapid eye movement (REM) sleep and ponto-geniculo-occipital (PGO) waves. Much of this work stems from systemic and central cholinergic drug administration studies. The brainstem cholinergic system is also implicated in cortical activation via basal forebrain, thalamic, and hypothalamic relay neurons. This cholinergic ascending reticular activating hypothesis has also been suggested by in vivo experiments under anesthetics and by in vitro studies using cholinergic agonists in thalamic and hypothalamic slices. During the last ten years, brainstem cholinergic neurons have been discovered to be in the peribrachial area (PBL). With the discovery of PBL cholinergic neurons, many studies were devoted to the examination of PBL neuronal activity and their connectivity. This article reviews PBL neuronal activity in behaving animals and the anatomical features of these neurons in relation to behavioral state control. The role of the PBL in the generation of REM sleep, PGO waves, and the ascending reticular activating system (ARAS) has been evaluated at the cellular and neurochemical level. Based on recent literature, tentative mechanisms of REM sleep generation, PGO waves generation, and the cortical activation process are also outlined.
The topographical relationships between cholinergic neurons, identified by their immunoreactivity for choline acetyltransferase (ChAT) or their staining for beta-nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, and dopaminergic, serotoninergic, noradrenergic, and glutamatergic neurons that occur in the mesopontine tegmentum, were studied in the squirrel monkey (Saimiri sciureus). The ChAT-positive neurons in the pedunculopontine nucleus (PPN) form two distinct subpopulations, one that corresponds to PPN pars compacta (PPNc) and the other to PPN pars dissipata (PPNd). The ChAT-positive neurons in PPNc are clustered along the dorsolateral border of the superior cerebellar peduncle (SP) at trochlear nucleus levels, whereas those in PPNd are scattered along the SP from midmesencephalic to midpontine levels. At levels caudal to the trochlear nucleus, ChAT-positive neurons corresponding to the laterodorsal tegmental nucleus (LDT) lie within the periaqueductal gray and extend caudally as far as locus coeruleus levels. All ChAT-positive neurons in PPN and LDT stain for NADPH-diaphorase; the majority of large neurons in PPN and LDT are cholinergic, but some large neurons devoid of NADPH-diaphorase also occur in these nuclei. Cholinergic neurons in the mesopontine tegmentum form clusters that are largely segregated from raphe serotonin-immunoreactive neurons, as well as from nigral dopaminergic and coeruleal noradrenergic neurons, as revealed by tyrosine hydroxylase immunohistochemistry. Nevertheless, dendrites of cholinergic and noradrenergic neurons are closely intermingled, suggesting the possibility of dendrodendritic contacts. In addition, numerous large and medium-sized glutamate-immunoreactive neurons are intermingled among cholinergic neurons in PPN. Furthermore, at trochlear nucleus levels, about 40% of cholinergic neurons display glutamate immunoreactivity, whereas other neurons express glutamate or ChAT immunoreactivity only. This study demonstrates that 1) cholinergic neurons remain largely segregated from monoaminergic neurons throughout the mesopontine tegmentum and 2) PPN contains cholinergic and glutamatergic neurons as well as neurons coexpressing ChAT and glutamate in primates.
Serotonergic synaptic inputs to cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei were examined with pre-embedding dual-label immunoelectron microscopy. Numerous serotonin-immunoreactive axon terminals visualized with a silver-enhanced immunogold method were present in both of these tegmental nuclei. Serotonergic terminals occasionally made synaptic contacts with the soma and proximal dendrites of cholinergic tegmental neurons labelled with a choline acetyltransferase-immunoreactive peroxidase-anti-peroxidase diaminobenzidine reaction product. In the rostralmost region of the laterodorsal tegmental nucleus, a few serotonergic neurons of the dorsal raphe nucleus were interspersed among cholinergic neurons. Some dendrites of these serotonergic neurons appeared to contain synaptic vesicles. Both myelinated and unmyelinated serotonergic axons were present in the mesopontine tegmentum. The presence of serotonergic synapses onto tegmental cholinergic neurons is consistent with previous behavioral and electrophysiological findings suggesting an inhibitory role of serotonin in the induction of rapid eye movement sleep and its phenomenology through an action on cholinergic neurons in the mesopontine tegmentum.
Intracellular recordings were obtained from neurons of the laterodorsal tegmental and pedunculopontine tegmental nuclei in a brain-slice preparation. The action of exogenously applied 5-hydroxytryptamine and acetylcholine was studied on NADPH-diaphorase-labeled cells which contain nitric oxide synthase and are presumed to be cholinergic. Our results indicated that these cells were hyperpolarized by both 5-hydroxytryptamine and acetylcholine; the ionic mechanism of this inhibition was investigated using current and voltage clamp methods. Cells voltage-clamped at resting membrane potential exhibited a net outward current and an increased membrane conductance during 5-hydroxytryptamine and acetylcholine mediated inhibition. The membrane hyperpolarization and outward current generated by this paradigm reversed near the expected K equilibrium potential and was blocked by low concentrations of extracellular Ba. The 5-hydroxytryptamine- and acetylcholine-dependent currents showed inward rectification and the reversal potential shifted in the depolarizing direction by about 15 mV for a doubling of extracellular K, indicating that both 5-hydroxytryptamine and acetylcholine activate inwardly rectifying, potassium-selective conductances. The 5-hydroxytryptamine-evoked hyperpolarization was antagonized by spiperone and mimicked by (+)8-hydroxy-2-(Di-N-propylamino)-tetralin suggesting the presence of a 5-hydroxytryptamine1A receptor while the acetylcholine-evoked hyperpolarization was blocked by atropine and only high concentrations of pirenzepine, suggesting a muscarinic M2 receptor. The outward currents evoked by 5-hydroxytryptamine and acetylcholine were not additive, suggesting that both receptors are coupled to an overlapping pool of K channels as has been observed in several systems in which receptors are coupled to effectors by G-proteins. These results indicate that the dominant actions of 5-hydroxytryptamine and acetylcholine relate to the inhibition of mesopontine cholinergic neurons via activation of an overlapping pool of inwardly rectifying K channels. Cholinergic neurons of these nuclei are thought to play an instrumental role in the induction and maintenance of rapid eye movement sleep. It has been previously hypothesized that acetylcholine would be excitatory and that 5-hydroxytryptamine would be inhibitory to these cells in the context of rapid eye movement sleep. [McCarley R. and Massaquoi S. (1986) Am. J. Physiol. 251, R1011-R1029; McCarley R. W. et al. (1975) Science 189, 58-60]. Our results are consistent with the proposed inhibitory action of 5-hydroxytryptamine but indicate recurrent input to cholinergic neurons would be inhibitory. Accordingly, models of the neural substrate underlying rapid eye movement sleep production need to be changed to reflect this inhibitory action of acetylcholine on cholinergic neurons.
The effects of brainstem microinjections of carbachol on the hippocampal theta rhythm were examined in urethane anesthetized rats. The two most effective theta-eliciting sites with carbachol were the nucleus pontis oralis (RPO) and the acetylcholine-containing pedunculopontine tegmental nucleus (PPT) of the dorsolateral pontine tegmentum. RPO injections generated theta at mean latencies of 38.5 +/- 70.8 s and for mean durations of 12.9 +/- 5.1 min. Five of seven RPO injections gave rise to theta virtually instantaneously, i.e., before the completion of the injection. PPT injections generated theta at mean latencies of 1.7 +/- 1.1 min and for mean durations of 11.9 +/- 6.0 min. Injections rostral or caudal to RPO in the caudal midbrain reticular formation (RF) or the caudal pontine RF (nucleus pontis caudalis) generated theta at considerably longer latencies (generally greater than 5 min) or were without effect. Medullary RF injections essentially failed to alter the hippocampal EEG. The finding that theta was produced at very short latencies at RPO suggests that RPO, the putative brainstem source for the generation of theta, is modulated by a cholinergic input. The further demonstration that theta was also very effectively elicited with PPT injections suggests this acetylcholine-containing nucleus of the dorsolateral pons may be a primary source of cholinergic input to RPO in the generation of theta. The hippocampal theta rhythm is a major event of REM sleep. The present results are consistent with earlier work showing that each of the other major events of REM sleep, as well as the REM state, are cholinergically activated at the level of the pontine tegmentum.
Synaptic connectivity and other ultrastructural features of cholinergic and non-cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei were investigated with electron microscopy combined with pre-embedding immunohistochemistry for choline acetyltransferase. Quantitative morphometric analyses were conducted on selected immunopositive as well as immunonegative neurons. The ultrastructure of immunoreactive neurons in the laterodorsal and pedunculopontine tegmental nuclei was similar. In both nuclei, immunoreactive neurons were among the larger neurons, and somatic areas of immunopositive neurons in single thin sections were larger than those of immunonegative neurons by an average of 40%. Immunopositive somata varied in shape, appearing polygonal, fusiform or oval. Regardless of immunoreactivity, however, neurons in the pedunculopontine nucleus tended to have more irregular shapes than those in the laterodorsal tegmental nucleus. Immunoreactive neurons in both the nuclei had abundant cytoplasmic organelles and a large, clear nucleus with a few infoldings. Usually, about a quarter of the surface of an immunopositive soma was covered with astrocytic processes, and some immunopositive somata were directly apposed to an astrocyte. Immunoreactive dendrites and, less frequently, axon terminals were seen in close apposition to endothelial cells of blood capillaries or pericytes. Immunoreactive somata and dendrites in the laterodorsal and pedunculopontine tegmental nuclei received many synapses, mainly from unlabelled axon terminals. The mean number (4.7 +/- 1.8) of synapses received by immunolabelled somata in single thin sections was greater, by about 70%, than those received by unlabelled somata. The presynaptic axon terminals synapsing with immunoreactive somata commonly contained small, round and clear vesicles, and 20% of them contained a few dense-cored vesicles as well. Immunoreactive dendrites, in addition, received synapses from unlabelled axon terminals containing flat and clear vesicles, which accounted for 15% of the synapses with immunoreactive dendrites. Many immunopositive axon terminals were present in both the tegmental nuclei. They contained clear round vesicles, and usually synapsed with unlabelled dendrites. A few immunolabelled axons, however, appeared to synapse with immunopositive somata and dendrites. Immunoreactive fibres were also present in both the tegmental nuclei. They were either thinly myelinated or unmyelinated. In conclusion, the ultrastructural morphology of cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei is similar, and these neurons represent a distinct population of neurons in both nuclei in that they are larger and receive more synaptic contacts than non-cholinergic neurons. Cholinergic neurons, however, appear to receive synapses from cholinergic axon terminals only rarely, despite the abundance of cholinergic terminals in the tegmental nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)
The serotonergic dorsal raphe nucleus is considered an important modulator of state-dependent neural activity via projections to cholinergic neurons of the pedunculopontine tegmental nucleus (PPT). Light and electron microscopic analysis of anterogradely transported biotinylated dextran, combined with choline acetyltransferase (ChAT) immunohistochemistry, were employed to describe the synaptic organization of mesopontine projections from the dorsal raphe to the PPT. In a separate set of experiments, we utilized immunohistochemistry for the serotonin transporter (SERT), combined with ChAT immunohistochemistry at the light and electron microscopic levels, to determine whether PPT neurons receive serotonergic innervation. The results of these studies indicate that: (1) anterogradely labeled and SERT-immunoreactive axons and presumptive boutons invest the PPT at the light microscopic level; (2) at the ultrastructural level, dorsal raphe terminals in the PPT pars compacta synapse mainly with dendrites and axosomatic contacts were not observed; (3) approximately 12% of dorsal raphe terminals synapse with ChAT-immunoreactive dendrites; and (4) at least 2-4% of the total synaptic input to ChAT-immunoreactive dendrites is of dorsal raphe and/or serotonergic origin. This serotonergic dorsal raphe innervation may modulate cholinergic PPT neurons during alterations in behavioral state. The role of these projections in the initiation of rapid eye movement (REM) sleep and the ponto-geniculo-occipital waves that precede and accompany REM sleep is discussed.
1. Pontogeniculooccipital (PGO) waves are recorded during rapid eye movement (REM) sleep from the pontine reticular formation. 2. PGO wave-like field potentials can also be recorded in many other parts of the brain in addition to the pontine reticular formation, but their distribution is different in different species. Species differences are due to variation in species-specific postsynaptic target sites of the pontine PGO generator. 3. The triggering neurons of the pontine PGO wave generator are located within the caudolateral peribrachial and the locus subceruleus areas. 4. The transferring neurons of the pontine PGO generator are located within the cholinergic neurons of the laterodorsal tegmentum and the pedunculopontine tegmentum. 5. The triggering and transferring neurons of the pontine PGO wave generator are modulated by aminergic, cholinergic, nitroxergic, GABA-ergic, and glycinergic cells of the brainstem. The PGO system is also modulated by suprachiasmatic, amygdaloid, vestibular, and brainstem auditory cell groups.
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).
The present review has provided evidence that very potent ascending brainstem hippocampal synchronizing pathways originate in the rostral pons region (RPO and PPT), and ascend to and synapse with several midline caudal diencephalic nuclei (posterior hypothalamic and supramammillary) which send projections to the medial septal region (MS/vDBB). The medial septal region in turn is a critical nodal point, sending projections to limbic structures such as the hippocampal formation, cingulate cortex, and entorhinal cortex. The pontine and diencephalic nuclei appear to play a critical role in determining the translation of increasing levels of activation into moment to moment changes in the frequency of hippocampal theta field and theta-related cellular discharges, relayed to the MS/vDBB nuclei. The MS/vDBB nuclei appear to play a critical role in translating increasing levels of ascending synchronizing activation into moment to moment changes in the amplitude of hippocampal theta field activity and the accompanying rate and pattern of phasic theta-ON cells. The MS/vDBB carries out this role through a balance of activity in the septohippocampal cholinergic and GABA-ergic projections. Cholinergic projections provide the afferent excitatory drive for hippocampal theta-ON cells and the GABA-ergic projections act to reduce the overall level of inhibition by inhibiting hippocampal GABA-ergic interneurons (theta-OFF cells). Both activities must be present for the generation of hippocampal theta and theta-related cellular activities. The balance between the cholinergic and GABA-ergic projections may determine whether hippocampal synchrony (theta) or asynchrony (LIA, large amplitude irregular activity) occurs. These same ascending pathways influence the electrophysiological and pharmacological properties of the neocortex as well. The functional significance of the ascending brainstem synchronizing pathways is the generalized regulation of activities in these cortical structures as they relate to sensorimotor behavior.
The substantia nigra (SN) has long been known as an important source of afferents to the pedunculopontine tegmental nucleus (PPN). However, it has not been established which of the chemospecific cell populations receive this synaptic input. We sought to address this issue by a correlative light and electron microscopic approach that combines anterograde tracing of nigral efferents with pre-embedding choline acetyltransferase (ChAT) and/or glutamate (Glu) immunohistochemistry. Following large bilateral injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) in the SN, the labeled nigrotegmental fibers were concentrated in a small area of the mesopontine tegmentum which contained very few ChAT-immunoreactive (ChAT-ir) cell bodies. However, strands of fine varicose fibers penetrated to adjacent regions of the PPN which harbored numerous cholinergic perikarya. The anterogradely labeled boutons were often seen in the proximity of ChAT-ir perikarya and dendrites, but the majority (82-93%) established symmetric synaptic junctions with noncholinergic profiles. In the pars dissipata of the PPN (PPNd), one-third of the labeled terminals synapsed onto noncholinergic perikarya and primary dendrites, while in the pars compacta of the PPN (PPNc) axosomatic synapses were rare. The possibility that the perikarya receiving a rich synaptic input from the SN are glutamatergic was tested in experiments combining anterograde transport of biotinylated tracers biocytin and dextran-amine (BDA) with glutamate immunohistochemistry. In double-labeled sections, Glu-ir perikarya within the terminal plexus of nigrotegmental fibers were surrounded by synaptic terminals. The PPNd also contained retrogradely BDA-labeled neurons which were contacted by anterogradely labeled terminals. These results indicate that although a small subpopulation of cholinergic neurons in the mesopontine tegmentum receive direct synaptic input from the SN, the primary target of nigrotegmental fibers are glutamatergic cells in the PPNd. Our results also provide ultrastructural evidence that some nigrotegmental fibers innervate pedunculonigral neurons.
Cholinergic neurons of the laterodorsal tegmental nucleus have been hypothesized to play a critical role in the generation and maintenance of rapid eye movement sleep. Less is known about the function of non-cholinergic laterodorsal tegmental nucleus neurons. As part of our ongoing studies of the brainstem circuitry controlling behavioral state, we have begun to investigate the functional properties of these neurons. In the course of these experiments, we have observed a novel response to the neurotransmitter noradrenaline. Whole-cell patch-clamp recordings of laterodorsal tegmental nucleus neurons were carried out in 21- to 35-day-old rat brain slices. A subpopulation of laterodorsal tegmental nucleus cells responded to a 30-s application of 50 μM noradrenaline with depolarization and a decrease in input resistance which lasted several minutes. Following return to resting membrane potential, these cells invariably exhibited barrages of excitatory postsynaptic potentials which lasted at least 12 min. These excitatory postsynaptic potentials were reversibly abolished by bath application of tetrodotoxin, as well as by the non-N-methyl-d-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, but were insensitive to application of the N-methyl-d-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid. To examine whether these neurons were cholinergic, the recorded cells were labeled with biocytin and tested for co-localization with reduced nicotinamide adenine dinucleotide phosphate-diaphorase, a marker for laterodorsal tegmental nucleus cholinergic neurons. In every instance, neurons with these properties were non-cholinergic. However, they were always located in close proximity to reduced nicotinamide adenine dinucleotide phosphate-diaphorase-positive laterodorsal tegmental nucleus cells.
The aim of this study was to test the hypothesis that the cells in the brain stem pedunculopontine tegmentum (PPT) are critically involved in the normal regulation of wakefulness and rapid eye movement (REM) sleep. To test this hypothesis, one of four different doses of the excitatory amino acid L-glutamate (15, 30, 60, and 90 ng) or saline (control vehicle) was microinjected unilaterally into the PPT while the effects on wakefulness and sleep were quantified in freely moving chronically instrumented rats. All microinjections were made during wakefulness and were followed by 6 h of polygraphic recording. Microinjection of 15- ng (0.08 nmol) and 30-ng (0.16 nmol) doses of L-glutamate into the PPT increased the total amount of REM sleep. Both doses of L-glutamate increased REM sleep at the expense of slow-wave sleep (SWS) but not wakefulness. Interestingly, the 60-ng (0.32 nmol) dose of L-glutamate increased both REM sleep and wakefulness. The total increase in REM sleep after the 60-ng dose of L-glutamate was significantly less than the increase from the 30-ng dose. The 90-ng (0.48 nmol) dose of L-glutamate kept animals awake for 2-3 h by eliminating both SWS and REM sleep. These results show that the L-glutamate microinjection into the PPT can increase wakefulness and/or REM sleep depending on the dosage. These findings support the hypothesis that excitation of the PPT cells is causal to the generation of wakefulness and REM sleep in the rat. In addition, the results of this study led to the identification of the PPT dosage of L-glutamate that optimally induces wakefulness and REM sleep. The knowledge of this optimal dose will be useful in future studies investigating the second messenger systems involved in the regulation of wakefulness and REM sleep.
Microinjection of the excitatory amino acid, L-glutamate into the brainstem pedunculo pontine tegmentum (PPT) has been shown to induce wakefulness, however, it has been unclear that receptors mediate this effect. The aim of this study was to test the hypothesis that in the PPT, L-glutamate induces cortical activation and wakefulness via activation of NMDA receptors. To test this hypothesis, three sets of micro-injections into the PPT were carried out on two different groups of rats that were then allowed to move freely although chronic instrumentation recorded sleep/wake states. Three days after the initial control injections of saline, in a contra-lateral site, Group I was micro-injected with saline + glutamate (saline first, and glutamate 15 min later); after another 3 days, the same rats were micro-injected with the NMDA-receptor-specific antagonist, 2-amino-5-phosphonopentanoic acid, (AP5) + glutamate. Group II received the same initial control injections (saline only), then AP5 + glutamate and the saline + glutamate micro-injections last. In rats that were not pretreated with AP5, microinjection of a 90 ng dose of L-glutamate (0.48 nmol in a volume of 0.1 microl vehicle) kept animals awake for 2-3 hr by eliminating both slow-wave sleep (SWS) and rapid eye movement (REM) sleep. These behavioral state changes were accompanied by concomitant increase in the power of gamma (gamma) frequency (20-60 Hz) waves in the cortical EEG. Pretreatment of L-glutamate injection sites with 0.48 nmol of AP5 blocked L-glutamate-induced-wakefulness and preserved a normal amount of wakefulness and sleep. Pretreatment with AP5 decreased the power of gamma-wave activity below its control level. These results support the hypothesis that the glutamate-induced-wakefulness and cortical activation effects are mediated via the NMDA receptors.
Ketamine induces a dissociated state of consciousness by binding to the phencyclidine binding site within the ion channel gated by the N-methyl-D-aspartate (NMDA) receptor. The brain regions and neurotransmitter systems mediating ketamine-induced alterations in arousal remain incompletely understood. This study used in vivo microdialysis to test the hypothesis that ketamine alters acetylcholine (ACh) release in the medial pontine reticular formation (mPRF).
Acetylcholine (ACh) release, sleep, and breathing were quantified following systemic ketamine administration. Microdialysis was used to deliver the NMDA-channel blocker dizocilpine maleate (MK-801) and the R(-)-isomer of ketamine into the mPRF while measuring ACh release.
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Systemically administered ketamine disrupted normal sleep-cycle organization, reduced mPRF ACh release, and significantly slowed rate of breathing. Dialysis delivery of MK-801 to the mPRF significantly decreased respiratory rate and mPRF ACh release. Dialysis delivery to the mPRF of the R(-)-ketamine isomer significantly decreased mPRF ACh release.
Decreased mPRF ACh release caused by systemically administered ketamine was mimicked by mPRF dialysis delivery of MK-801 and the R(-)-ketamine isomer. These data are consistent with the conclusion that systemically administered ketamine may alter arousal and breathing, in part, by altering cholinergic neurotransmission in the mPRF.
Microinjections of the excitatory amino acid, L-glutamate into the cholinergic cell compartment of the pedunculopontine tegmentum (PPT) of the rat induces both wakefulness and/or rapid eye movement (REM) sleep depending on the glutamate dosage. However, no studies have systematically recorded the electrical activity of these cells in the freely moving rat across the sleep-wake cycle. In this study, we have recorded the spontaneous activity patterns of single PPT cells (n = 70) in the freely moving rat across the sleep-wake cycle. PPT neurons were classified into three groups based on patterns in their spontaneous activity. The first group of cells (12.86%) was more active during REM sleep than they were during wakefulness or slow-wave sleep (SWS). The second group of cells (60.0%) was more active during REM and wakefulness than during SWS. The firing rate of the third group of cells (27.14%) did not change as a function of behavioral state. This study also demonstrated that the level of activity within the cholinergic cell compartment of the PPT during SWS drops to 7.4% of that observed during wakefulness and that during REM sleep it changes to 65.5% of wakefulness levels. These findings indicate that in the freely moving rat, the discharging of PPT neurons correlates with wakefulness and REM sleep. Additionally, these neurons may be an integral part of the brainstem wakefulness and REM sleep-generating mechanisms in the rat.
The regional organization of the ventral respiratory group (VRG) was examined with respect to generation of respiratory rhythm (breathing frequency) versus control of the respiratory motor pattern on individual nerves. In urethane-anaesthetized, neuromuscularly blocked and vagotomized Sprague-Dawley rats, arterial blood pressure (ABP) and respiratory motor outputs (phrenic, pharyngeal branch of the vagus, or superior laryngeal nerves) were recorded. The VRG organization was mapped systematically using injections of the excitatory amino acid DL-homocysteic acid (DLH; 5-20 mM, 2-6 nl) from single- or double-barrel pipettes at 100-200 microm intervals between the facial nucleus and the calamus scriptorius. Recording of respiratory neurons through the injection pipette ensured that the pipette was located within the VRG. At the end of each experiment, the injection pipette was used to make an electrical lesion, thereby marking the electrode position for subsequent histological reconstruction of injection sites. Four rostrocaudal regions were identified: (1) a rostral bradypnoea area, at the level of the Bötzinger complex, in which respiratory rhythm slowed and ABP increased, (2) a tachypnoea/dysrhythmia area, at the level of the preBötzinger complex, in which breathing rate either increased or became irregular, with little or no change in ABP, (3) a caudal bradypnoea area at the level of the anterior part of the rostral VRG in which ABP decreased and (4) a caudal 'no effect' region in the posterior part of the rostral VRG. The peak amplitude of phrenic nerve activity decreased with injections into all three rostral regions. Changes in respiratory rhythm were associated with opposite changes in inspiratory (TI) and expiratory (TE) durations after injections into either the Bötzinger complex or anterior rostral VRG, while both TI and TE decreased after injections into the preBötzinger complex. Effects on selected cranial nerves were similar to those on the phrenic nerve except that tonic activity was elicited on the superior larygneal nerve ipsilateral to injections in the Bötzinger complex and on the pharyngeal branch of the vagus ipsilateral to injections in the preBötzinger complex. These data reinforce the subdivision of the VRG into functionally distinct compartments and suggest that a further subdivision of the rostral VRG is warranted. They also suggest that region-specific influences, especially on the pattern of cranial motor discharge, can be used to assist the identification of recording sites within the VRG. However, the postulated clear functional separation of rhythm- versus pattern-generating regions was not supported.
Considerable evidence suggests that rapid eye movement (REM) sleep is induced by glutamatergic activation of cholinergic cells within the pedunculopontine tegmentum (PPT). The aim of this study is to test a popular hypothesis that serotonin, norepinephrine, and adenosine act on PPT cells to regulate REM sleep. This study also tests an alternate hypothesis that serotonin may inhibit REM sleep signs by direct action on the individual REM sleep sign generators.
Serotonin, norepinephrine, and adenosine were locally microinjected into the PPT and serotonin was microinjected into the pontine-wave (P-wave) generator (dorsal part of the locus subcoeruleus nucleus) while quantifying the effects on REM sleep and P-wave activity in freely moving rats.
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Local microinjections of serotonin, norepinephrine, and adenosine into the PPT did not change REM sleep. Microinjection of serotonin into the P-wave generator suppressed P-wave activity but not REM sleep.
The present findings provide direct evidence that serotonin, norepinephrine, and adenosine-induced REM sleep suppression in the behaving rat are not mediated by the PPT. The results also provide direct evidence, for the first time, that serotonin suppresses P-wave activity by acting directly on the P-wave generator. These results suggest that the serotonin-induced inhibition of REM sleep in the freely moving rat is probably not mediated through the mesopontine cholinergic cell compartment but, rather, through individual REM sleep sign generators.
This study demonstrates respiratory modulation caused by stimulation of the pedunculopontine tegmental nucleus (PPT), a structure not classically included in the pontine respiratory neuronal network. The long-lasting increase in variability of respiratory parameters following glutamate microinjection into PPT in anesthetized, spontaneously breathing Sprague Dawley rats was more pronounced under ketamine than nembutal anesthesia. The induced respiratory perturbations were characterized by intermittent apneas and increased variability of expiratory (TE) and total (TT) breath durations in all animals. Although the baseline spontaneous breathing patterns (mean values of all respiratory parameters and their variabilities) were equivalent under ketamine and nembutal anesthesia, different anesthetic agents did affect respiratory responses to PPT stimulation by glutamate in terms of latency, duration, and structure. We conclude that glutamatergic stimulation of PPT has a significant impact on the brainstem respiratory pattern generator.
We describe and summarize here our recent findings about the role in respiration of two pontine structures that are not classically included in the pontine respiratory group: the pedunculopontine tegmental nucleus (PPT) and the intertrigeminal region (ITR). We also discuss significant contributions of other workers in the field, especially, S. Datta [Cell. Mol. Neurobiol. 17: 341-365, 1997], R. Lydic and H. Baghdoyan [Sleep, 25: 617-622, 2002], and N. Chamberlin and C. Saper [J. Neurosci. 18: 6048-6056, 1998], who postulated a role for the ITR in modulating reflex apnea. In anesthetized and freely moving rats we have consistently documented that PPT and ITR have a role in respiration. Neurochemical manipulations of each area affected the brainstem respiratory pattern generator and respiratory pattern variability,observed as spontaneous disturbances during sleep or as induced reflex apnea. Although the exact central mechanisms of apnea cannot be determined from our studies to date, we postulate that reflex and sleep-related apneas in rats share some common brainstem pathways, which may include PPT and ITR.
Activation of pontomedullary cholinergic neurons may directly and indirectly cause depression of respiratory motoneuronal activity, activation of respiratory premotor neurons and acceleration of the respiratory rate during REM sleep, as well as activation of breathing during active wakefulness. These effects may be mediated by distinct subpopulations of cholinergic neurons. The relative inactivity of cholinergic neurons during slow-wave sleep also may contribute to the depressant effects of this state on breathing. Cholinergic muscarinic and nicotinic receptors are expressed in central respiratory neurons and motoneurons, thus allowing cholinergic neurons to act on the respiratory system directly. Additional effects of cholinergic activation are mediated indirectly by noradrenergic, serotonergic and other neurons of the reticular formation. Excitatory and suppressant respiratory effects with features of natural states of REM sleep or active wakefulness can be elicited in urethane-anesthetized rats by pontine microinjections of the cholinergic agonist, carbachol. Carbachol models help elucidate the neural basis of respiratory disorders associated with central cholinergic activation.
Cholinergic pathways ascending from the brainstem are considered as a decisive part of the reticular activating system. We recorded unit activity from the cholinergic pedunculopontine tegmental nucleus with extracellular microelectrodes in urethane-anesthetized rats and monitored cortical electroencephalogram (EEG) to examine the possible role of the nucleus in cortical activation. We found two types of cells showing EEG-correlated firing patterns. In one group, firing rate increased during cortical activation (F cell), while in another, higher rate was accompanied by cortical slow waves (S cell). Phasic changes in the firing rate of pedunculopontine neurons and in the cortical EEG were also analyzed. Changes of single unit activity in F cells always occurred before short periods of low-voltage fast activity appeared in the cortical EEG. The S cells were more variable with respect to the temporal relation. In some of the S cells, changes in firing rate preceded changes in the EEG patterns, while in others they occurred after a certain delay. Our results indicate that F-cells in the PPT might be involved in the initiation of tonic and phasic changes in cortical activation.