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Mesopontine cholinergic projections to the hypoglossal motor nucleus
Abstract
Mesopontine cholinergic (ACh) neurons have increased discharge during wakefulness, rapid eye movement (REM) sleep, or both. Hypoglossal (12) motoneurons, which play an important role in the control of upper airway patency, are postsynaptically excited by stimulation of nicotinic receptors, whereas muscarinic receptors presynaptically inhibit inputs to 12 motoneurons. These data suggest that ACh contributes to sleep/wake-related changes in the activity of 12 motoneurons by acting within the hypoglossal motor nucleus (Mo12), but the origins of ACh projections to Mo12 are not well established. We used retrograde tracers to assess the projections of ACh neurons of the mesopontine pedinculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei to the Mo12. In six Sprague-Dawley rats, Fluorogold or B subunit of cholera toxin, were pressure injected (5-20nl) into the Mo12. Retrogradely labeled neurons, identified as ACh using nitric oxide synthase (NOS) immunohistochemistry, were found bilaterally in discrete subregions of both PPT and LDT nuclei. Most retrogradely labeled PPT cells (96%) were located in the PPT pars compacta region adjacent to the ventrolateral tip of the superior cerebellar peduncle. In the LDT, retrogradely labeled neurons were located exclusively in its pars alpha region. Over twice as many ACh neurons projecting to the Mo12 were located in the PPT than LDT. The results demonstrate direct mesopontine ACh projections to the Mo12. These projections may contribute to the characteristic of wakefulness and REM sleep increases, as well as REM sleep-related decrements, of 12 motoneuronal activity.
... In addition to connections with other components of the REM sleep control network already described; PPTn neurons innervate critical components of the respiratory network. PPTn neurons project to areas of the rostral ventrolateral medulla (Yasui et al., 1990) that contain respiratory neurons critical to the generation of respiratory rhythm and pattern (Feldman and Del Negro, 2006), and to relevant motor pools such as the hypoglossal motor nucleus (Woolf and Butcher, 1989;Fay and Norgren, 1997;Rukhadze and Kubin, 2007), which innervates the genioglossus muscle of the tongue. or having no effect in many cases (Bourgin et al., 1995;Deurveilher et al., 1997;Boissard et al., 2002;Pollock and Mistlberger, 2005) and endogenous acetylcholine in the PRF has not yet been shown to be necessary for REM sleep generation as would be tested by focal application of acetylcholine receptor antagonists. ...
... Likewise, GABA A receptor-mediated inhibition of PPTn neurons results in increased REM sleep (Torterolo et al., 2002;Mallick, 2004, 2009 (Thakkar et al., 1998) will show that this cell group functions to suppress REM sleep and its phenomenological components. For the latter I measured indices of respiratory network activity, given the projections of PPTn to the critical brainstem sites modulating respiratory rhythm and motor activities (Woolf and Butcher, 1989;Yasui et al., 1990;Fay and Norgren, 1997;Rukhadze and Kubin, 2007) ...
... thalamus (Semba et al., 1990;Semba and Fibiger, 1992;Bevan and Bolam, 1995), and so are thought to modulate REM sleep and the associated electrocortical activation . Neurons in this sub-region of the PPTn also project to the areas of the rostral ventrolateral medulla (Yasui et al., 1990) that contain respiratory neurons critical to the generation of respiratory rhythm and pattern (Feldman and Del Negro, 2006), and to relevant motor pools such as the hypoglossal motor nucleus which innervates the genioglossus muscle of the tongue (Woolf and Butcher, 1989;Fay and Norgren, 1997;Rukhadze and Kubin, 2007), relaxation of which is instrumental to the pathogenesis of obstructive sleep apnea (Remmers et al., 1978). ...
... Nevertheless, remarkable are the efferent bilateral connections to the medullary region, especially to the rostroventrolateral medulla, the caudal half of the NTS and the dorsal motor nucleus of the vagus [28,[40][41][42]. They also show that the innervation is completely noradrenergic and that more than half of the neurones in area A5 project ipsilaterally to both nuclei, but in some cases, the projection could be bilateral. ...
... Other work has described efferent connections from area A5 to the retrotrapezoid nucleus [34], an area that has been suggested to be the location of central chemoreceptors [46]. In another work, it was described that the motor nucleus of the hypoglossus and the NTS viscerosensory nucleus receive efferent connections from area A5 in a proportion of 46% and 43% of the total catecholaminergic projections they receive, respectively, suggesting that area A5 may play an important role in the modulation of motor flow and viscerosensory transmission [41]. ...
Area A5 is a noradrenergic cell group in the brain stem characterised by its important role in triggering sympathetic activity, exerting a profound influence on the sympathetic outflow, which is instrumental in the modulation of cardiovascular functions, stress responses and various other physiological processes that are crucial for adaptation and survival mechanisms. Understanding the role of area A5, therefore, not only provides insights into the basic functioning of the sympathetic nervous system but also sheds light on the neuronal basis of a number of autonomic responses. In this review, we look deeper into the specifics of area A5, exploring its anatomical connections, its neurochemical properties and the mechanisms by which it influences sympathetic nervous system activity and cardiorespiratory regulation and, thus, contributes to the overall dynamics of the autonomic function in regulating body homeostasis.
... Muscarinic antagonists in the MoXII reactivate the genioglossus muscle in REM sleep supporting a cholinergic-mediated depression of genioglossus activity in REM sleep (Grace et al., 2013). There are two main sources of cholinergic afferent input to the MoXII: the pedunculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei and cholinergic neurons in the IRt region (Travers et al., 2005;Rukhadze and Kubin, 2007;Volgin et al., 2008). LDT/PPT cholinergic neurons fire in REM sleep, suggesting that they could be the source of REM sleep-related cholinergic inhibition to HMNs (Boucetta et al., 2014), although the density of cholinergic innervation they provide to the MoXII is small (Rukhadze and Kubin, 2007). ...
... There are two main sources of cholinergic afferent input to the MoXII: the pedunculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei and cholinergic neurons in the IRt region (Travers et al., 2005;Rukhadze and Kubin, 2007;Volgin et al., 2008). LDT/PPT cholinergic neurons fire in REM sleep, suggesting that they could be the source of REM sleep-related cholinergic inhibition to HMNs (Boucetta et al., 2014), although the density of cholinergic innervation they provide to the MoXII is small (Rukhadze and Kubin, 2007). Conversely, IRt cholinergic neurons provide a denser input to the MoXII (Travers et al., 2005;Volgin et al., 2008), but their physiological role has yet to be demonstrated. ...
Proper function of pharyngeal dilator muscles, including the genioglossus muscle of the tongue, is required to maintain upper airway patency. During sleep, the activity of these muscles is suppressed, and as a result individuals with obstructive sleep apnea experience repeated episodes of upper airway closure when they are asleep, in particular during rapid-eye-movement (REM) sleep. Blocking cholinergic transmission in the hypoglossal motor nucleus (MoXII) restores REM sleep genioglossus activity, highlighting the importance of cholinergic transmission in the inhibition of hypoglossal motor neurons (HMNs) during REM sleep. Glutamatergic afferent input from neurons in the parahypoglossal (PH) region to the HMNs is critical for MoXII respiratory motor output. We hypothesized that state-dependent cholinergic regulation may be mediated by this pathway. Here we studied the effects of cholinergic transmission in HMNs in adult male and female mice using patch-clamp recordings in brain slices. Using channelrhodopsin-2-assisted circuit mapping, we first demonstrated that PH glutamatergic neurons directly and robustly activate HMNs (PHGlut → HMNs). We then show that carbachol consistently depresses this input and that this effect is presynaptic. Additionally, carbachol directly affects HMNs by a variable combination of muscarinic-mediated excitatory and inhibitory responses. Altogether, our results suggest that cholinergic signaling impairs upper airway dilator muscle activity by suppressing glutamatergic input from PH premotoneurons to HMNs and by directly inhibiting HMNs. Our findings highlight the complexity of cholinergic control of HMNs at both the presynaptic and postsynaptic levels and provide a possible mechanism for REM sleep suppression of upper airway muscle activity.SIGNIFICANCE STATEMENT Individuals with obstructive sleep apnea can breathe adequately when awake but experience repeated episodes of upper airway closure when asleep, in particular during REM sleep. Similar to skeletal postural muscles, pharyngeal dilator muscles responsible for maintaining an open upper airway become hypotonic during REM sleep. Unlike spinal motoneurons controlling postural muscles that are inhibited by glycinergic transmission during REM sleep, hypoglossal motoneurons that control the upper airway muscles are inhibited in REM sleep by the combination of monoaminergic disfacilitation and cholinergic inhibition. In this study, we demonstrated how cholinergic signaling inhibits hypoglossal motoneurons through presynaptic and postsynaptic muscarinic receptors. Our results provide a potential mechanism for upper airway hypotonia during REM sleep.
... In our studies, we injected retrograde tracers, FluoroGold and Cholera toxin B subunit, into the hypoglossal motor nucleus by an air pressure-driven delivery system (79,80). In agreement with the earlier study of Woolf and Butcher (78), ∼1% of PPT/LDT cholinergic neurons projected to the hypoglossal motor nucleus. ...
... In agreement with the earlier study of Woolf and Butcher (78), ∼1% of PPT/LDT cholinergic neurons projected to the hypoglossal motor nucleus. However, the PPT/LDT projections to hypoglossal motoneurons were bilateral (79). In another study, we found that ∼40% of cholinergic neurons of the caudal IRt region in medulla projected to the hypoglossal nucleus (80). ...
Obstructive Sleep Apnea (OSA) is a common sleep-related respiratory disorder that is associated with cognitive, cardiovascular, and metabolic morbidities. The major cause of OSA is the sleep-related reduction of upper airway muscle tone that leads to airway obstructions in individuals with anatomically narrow upper airway. This reduction is mainly due to the suppressant effect of sleep on hypoglossal motoneurons that innervate upper airway muscles. The hypoglossal motoneurons have state-dependent activity, which is decreased during the transition from wakefulness to non-rapid eye movement sleep and is further suppressed during rapid eye movement sleep. Multiple neurotransmitters and their receptors have been implicated in the control of hypoglossal motoneuron activity across the sleep-wake states. However, to date, the results of the rigorous testing show that withdrawal of noradrenergic excitation and cholinergic inhibition essentially contribute to the depression of hypoglossal motoneuron activity during sleep. The present review will focus on origins of noradrenergic and cholinergic innervation of hypoglossal motoneurons and the functional role of these neurons in the state-dependent activity of hypoglossal motoneurons.
... Evidence of projections in the reverse direction, from the PRG to the mPRF, is provided by microinjection of fluorescent dyes into the PRG, resulting in ipsi-and contralateral positive regions in the mPRF as well as in the dorsal respiratory group and ventral respiratory group in the medulla (20). However, studies of the connectivity between brain stem respiratory neurons performed using chronically implanted arrays of microelectrodes emphasized "a sparse distribution of functional connections," which suggests that interactions between brain stem neurons include less direct pathways (29,32). ...
... The cause of these effects has not been established. Since there are projections from pontine cholinergic neurons (32) to the hypoglossal nucleus, one potential contributing factor is altered cholinergic modulations of the motoneurons in the hypoglossal nucleus. Indeed, Kodama et al. (19) found in decerebrate cats that microinjection of ACh into the rostral pons causes GG muscle atonia while simultaneously increasing both glycine and GABA release in the hypoglossal nucleus. ...
For many years, acetylcholine has been known to contribute to the control of breathing and sleep. To probe further the contributions of cholinergic rostral pontine systems in control of breathing, we designed this study to test the hypothesis that microdialysis (MD) of the muscarinic receptor antagonist atropine into the pontine respiratory group (PRG) would decrease breathing more in animals while awake than while in NREM sleep. In 16 goats, cannulas were bilaterally implanted into rostral pontine tegmental nuclei (n = 3), the lateral (n = 3) or medial (n = 4) parabrachial nuclei, or the Kölliker-Fuse nucleus (KFN; n = 6). After >2 wk of recovery from surgery, the goats were studied during a 45-min period of MD with mock cerebrospinal fluid (mCSF), followed by at least 30 min of recovery and a second 45-min period of MD with atropine. Unilateral and bilateral MD studies were completed during the day and at night. MD of atropine into the KFN at night decreased pulmonary ventilation and breathing frequency and increased inspiratory and expiratory time by 12-14% during both wakefulness and NREM sleep. However, during daytime studies, MD of atropine into the KFN had no effect on these variables. Unilateral and bilateral nighttime MD of atropine into the KFN increased levels of NREM sleep by 63 and 365%, respectively. MD during the day or at night into the other three pontine sites had minimal effects on any variable studied. Finally, compared with MD of mCSF, bilateral MD of atropine decreased levels of acetylcholine and choline in the effluent dialysis fluid. Our data support the concept that the KFN is a significant contributor to cholinergically modulated control of breathing and sleep.
... Large motor neurons of the trigeminal nerve were particularly heavily innervated, however, by distinctly large VAChTϩ varicosities over their soma, which appeared different from those on reticular neurons. These varicosities likely comprise recurrent collaterals of the motor neurons, which are also known to be associated with M2Rs as autoreceptors (Hellstrom et al., 2003); however, could also arise in part, as for those in the hypoglossal nucleus (Rukhadze and Kubin, 2007), from LDT/PPT neurons projecting into this region (Jones, 1990). Acting on M2Rs, ACh released from terminals of LDT/PPT neurons could act to hyperpolarize and inhibit large reticular neurons, which in turn normally excite motor neurons, and thus to disfacilitate motor neurons in the spinal cord and/or brainstem. ...
... Acting on M2Rs, ACh released from terminals of LDT/PPT neurons could act to hyperpolarize and inhibit large reticular neurons, which in turn normally excite motor neurons, and thus to disfacilitate motor neurons in the spinal cord and/or brainstem. It could possibly also act more directly to inhibit motor neurons in the brainstem (Rukhadze and Kubin, 2007). In addition to large reticular neurons, VAChTϩ varicosities were also present on some small, GABAergic neurons in the reticular formation, particularly in the Mes. ...
Acetylcholine (ACh) plays an important role in the promotion of paradoxical sleep (PS) with muscle atonia through the muscarinic-2 receptor (M2R) in the mesopontine tegmentum. Conversely, orexin (Orx or hypocretin) appears to be critical for the maintenance of waking with muscle tone through the orexin-2 (or hypocretin-B) receptor (Orx2R), which is lacking in dogs having narcolepsy with cataplexy. In dual-immunostained material viewed under fluorescence microscopy, we examined the presence and distribution of M2R or Orx2R labeling on all neuronal nuclei (NeuN)-stained neurons or on glutamic acid decarboxylase (GAD)-stained neurons through the mesopontine tegmentum. Applying stereological analysis, we determined that many neurons bear M2Rs on their membrane ( approximately 6,300), including relatively large, non-GABAergic cells, which predominate (>75%) in the oral and caudal pontine (PnO and PnC) reticular fields, and small, GABAergic cells ( approximately 2,800), which predominate (>80%) in the mesencephalic (Mes) reticular formation. Many neurons bear Orx2Rs on their membrane ( approximately 6,800), including relatively large, non-GABAergic cells, which predominate (>70%) through all reticular fields, and comparatively few GABAergic cells ( approximately 700). In triple-immunostained material viewed by confocal microscopy, many large neurons in PnO and PnC appear to bear both M2Rs and Orx2Rs on their membrane, indicating that ACh and Orx could exert opposing influences of inhibition vs. excitation on putative reticulo-spinal neurons and thus attenuate vs. facilitate activity and muscle tone. A few GABAergic cells bear both receptors and could as PS inhibitor neurons serve under these different influences to control PS effector neurons and accordingly gate PS and muscle atonia appropriately across sleep-wake states.
... The source and operation of the cholinergic respiratory and sleep-related modulation of the HMN in vivo is currently unknown. There are two main groups of premotor cholinergic inputs to the HMN: (i) the intermediate reticular nucleus (IRt) in the lateral medulla, and (ii) the pedunculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei in the pons [59][60][61][62][63][64] . The IRt is the critical relay for the transmission of respiratory drive to the HMN and hypoglossal nerve in-vitro [65][66][67][68] . ...
Successful cholinergic-noradrenergic pharmacotherapy for obstructive sleep apnea (OSA) is thought to be due to effects at the hypoglossal motor nucleus (HMN). Clinical efficacy varies with muscarinic-receptor (MR) subtype affinities. We hypothesized that oxybutynin (cholinergic agent in successful OSA pharmacotherapy) is an effective MR antagonist at the HMN and characterized its efficacy with other antagonists. We recorded tongue muscle activity of isoflurane anesthetized rats (121 males and 60 females, 7–13 per group across 13 protocols) in response to HMN microperfusion with MR antagonists with and without: (i) eserine-induced increased endogenous acetylcholine at the HMN and (ii) muscarine. Eserine-induced increased acetylcholine decreased tongue motor activity (p < 0.001) with lesser cholinergic suppression in females versus males (p = 0.017). Motor suppression was significantly attenuated by the MR antagonists atropine, oxybutynin, and omadacycline (MR2 antagonist), each p < 0.001, with similar residual activity between agents (p ≥ 0.089) suggesting similar efficacy at the HMN. Sex differences remained with atropine and oxybutynin (p < 0.001 to 0.05) but not omadacycline (p = 0.722). Muscarine at the HMN also decreased motor activity (p < 0.001) but this was not sex-specific (p = 0.849). These findings have translational relevance to antimuscarinic agents in OSA pharmacotherapy and understanding potential sex differences in HMN suppression with increased endogenous acetylcholine related to sparing nicotinic excitation.
... Numerous cholinergic neurons are localized in the medullary reticular formation close to the ventral medullary area [39]. Hypoglossal nerve motoneurons [40], pre-Bötzinger complex (respiratory rhythm generator), and phrenic motoneurons receive cholinergic input [39]. ...
Despite the severe respiratory problems reducing the quality of life for Alzheimer's disease (AD) patients, their causes are poorly understood. We aimed to investigate hypoxic and hypercapnic respiratory responses in a transgenic mouse model of AD (AβPP V717I) overexpressing AβPP and mimicking early-onset AD. The cholinesterase inhibitor rivastigmine and the NMDA receptor antagonist memantine were used to investigate the effects of drugs, used to treat AD cognitive dysfunction, on breathing in hypoxia and hypercapnia. We found a significant increase in the respiratory response to hypercapnia and no difference in the hypoxic response in APP+ mice, compared with the control group (APP-). Memantine had no effect on respiration in either group, including responses to hypoxia and hypercapnia. Rivastigmine depressed resting ventilation and response to hypercapnia irrespective of the mice genotype. Reduction in hypoxia-augmented ventilation by rivastigmine was observed only in APP+ mice, which exhibited lower acetylcholinesterase activity in the hippocampus. Treatment with rivastigmine reduced the enzyme activity in both groups equally in the hippocampus and brainstem. The increased ventilatory response to hypercapnia in transgenic mice may indicate alterations in chemoreceptive respiratory nuclei, resulting in increased CO2 sensitivity. Rivastigmine is a potent reductant of normoxic and hypercapnic respiration in APP+ and APP- mice.
... Conventional approaches with non-specific tracers have been used to classify the major inputs from the brainstem to the 12N [12][13][14], while little attention has been paid to whole-brain inputs. Using horseradish peroxidase, a nonspecific tracer, a previous study showed that the 12N mainly receives inputs from brainstem reticular regions, the nucleus of the solitary tract (Sol), and the sensory trigeminal complex [14]. ...
Hypoglossal motor neurons (HMNs) innervate tongue muscles and play key roles in a variety of physiological functions, including swallowing, mastication, suckling, vocalization, and respiration. Dysfunction of HMNs is associated with several diseases, such as obstructive sleep apnea (OSA) and sudden infant death syndrome. OSA is a serious breathing disorder associated with the activity of HMNs during different sleep–wake states. Identifying the neural mechanisms by which the state-dependent activities of HMNs are controlled may be helpful in providing a theoretical basis for effective therapy for OSA. However, the presynaptic partners governing the activity of HMNs remain to be elucidated. In the present study, we used a cell-type-specific retrograde tracing system based on a modified rabies virus along with a Cre/loxP gene-expression strategy to map the whole-brain monosynaptic inputs to HMNs in mice. We identified 53 nuclei targeting HMNs from six brain regions: the amygdala, hypothalamus, midbrain, pons, medulla, and cerebellum. We discovered that GABAergic neurons in the central amygdaloid nucleus, as well as calretinin neurons in the parasubthalamic nucleus, sent monosynaptic projections to HMNs. In addition, HMNs received direct inputs from several regions associated with respiration, such as the pre-Botzinger complex, parabrachial nucleus, nucleus of the solitary tract, and hypothalamus. Some regions engaged in sleep–wake regulation (the parafacial zone, parabrachial nucleus, ventral medulla, sublaterodorsal tegmental nucleus, dorsal raphe nucleus, periaqueductal gray, and hypothalamus) also provided primary inputs to HMNs. These results contribute to further elucidating the neural circuits underlying disorders caused by the dysfunction of HMNs.
... Serotonergic innervation to XII motoneurons, which derives primarily from the caudal medullary raphe, primarily nuclei raphe pallidus and obscurus (Manaker & Tischler, 1993;Henry & Manaker, 1998), is sparse at P0 but increases until at least P28 . Cholinergic input to the XII nucleus originates from the laterodorsal and pedunculopontine tegmental nuclei, which are important for generation and maintenance of rapid eye movement (REM) sleep (Rukhadze & Kubin, 2007b). Additional sources include the reticular formation (Volgin et al. 2008) and C boutons, which are large cholinergic nerve terminals of unknown origin in the XII nucleus (Hellstrom et al. 2003), but in spinal motor nuclei are derived exclusively from Dbx1-and Pitx2-expressing interneurons (Zagoraiou et al. 2009). ...
Key points
Persistent inward currents (PICs) in spinal motoneurons are long‐lasting, voltage‐dependent currents that increase excitability; they are dramatically potentiated by serotonin, muscarine, and noradrenaline (norepinephrine).
Loss of these modulators (and the PIC) during sleep is hypothesized as a major contributor to REM sleep atonia.
Reduced excitability of XII motoneurons that drive airway muscles and maintain airway patency is causally implicated in obstructive sleep apnoea (OSA), but whether XII motoneurons possess a modulator‐sensitive PIC that could be a factor in the reduced airway tone of sleep is unknown.
Whole‐cell recordings from rat XII motoneurons in brain slices indicate that PIC amplitude increases ∼50% between 1 and 23 days of age, when potentiation of the PIC by 5HT2, muscarinic, or α1 noradrenergic agonists peaks at <50%, manyfold lower than the potentiation observed in spinal motoneurons.
α1 noradrenergic receptor activation produced changes in XII motoneuron firing behaviour consistent with PIC involvement, but indicators of strong PIC activation were never observed; in vivo experiments are needed to determine the role of the modulator‐sensitive PIC in sleep‐dependent reductions in airway tone.
Abstract
Hypoglossal (XII) motoneurons play a key role in maintaining airway patency; reductions in their excitability during sleep through inhibition and disfacilitation, i.e. loss of excitatory modulation, is implicated in obstructive sleep apnoea. In spinal motoneurons, 5HT2, muscarinic and α1 noradrenergic modulatory systems potentiate persistent inward currents (PICs) severalfold, dramatically increasing excitability. If the PICs in XII and spinal motoneurons are equally sensitive to modulation, loss of the PIC secondary to reduced modulatory tone during sleep could contribute to airway atonia. Modulatory systems also change developmentally. We therefore characterized developmental changes in magnitude of the XII motoneuron PIC and its sensitivity to modulation by comparing, in neonatal (P1–4) and juvenile (P14–23) rat brainstem slices, the PIC elicited by slow voltage ramps in the absence and presence of agonists for 5HT2, muscarinic, and α1 noradrenergic receptors. XII motoneuron PIC amplitude increased developmentally (from −195 ± 12 to −304 ± 19 pA). In neonatal XII motoneurons, the PIC was only potentiated by α1 receptor activation (5 ± 4%). In contrast, all modulators potentiated the juvenile XII motoneurons PIC (5HT2, 5 ± 5%; muscarine, 22 ± 11%; α1, 18 ± 5%). These data suggest that the influence of the PIC and its modulation on XII motoneuron excitability will increase with postnatal development. Notably, the modulator‐induced potentiation of the PIC in XII motoneurons was dramatically smaller than the 2‐ to 6‐fold potentiation reported for spinal motoneurons. In vivo measurements are required to determine if the modulator‐sensitive, XII motoneuron PIC is an important factor in sleep‐state dependent reductions in airway tone.
... ACh has long been known to play an important role in waking and PS or REMS, including muscle atonia as evidenced by severe reduction or elimination of PS following lesions of the LDT/PPT cholinergic neurons (Webster and Jones 1988) and induction of PS or REMS by administration of cholinergic agonists into the pontine reticular formation, an effect which is dependent upon AChM2Rs (Baghdoyan and Lydic 1999;Velazquez-Moctezuma et al. 1989). ACh can apparently play this role in part through direct influence on motor neurons, which for the Mo5 and Mo12, have been shown to receive input from cholinergic neurons of the LDT and pedunculopontine tegmental (PPT) nuclei and of the medullary reticular formation (Jones 1990;Woolf and Butcher 1989;Rukhadze and Kubin 2007;Fort et al. 1990). Cholinergic neurons of LDT/PPT discharge during waking and PS and are silent during SWS (Boucetta et al. 2014). ...
Muscle tone is regulated across sleep-wake states, being maximal in waking, reduced in slow wave sleep (SWS) and absent in paradoxical or REM sleep (PS or REMS). Such changes in tone have been recorded in the masseter muscles and shown to correspond to changes in activity and polarization of the trigeminal motor 5 (Mo5) neurons. The muscle hypotonia and atonia during sleep depend in part on GABA acting upon both GABAA and GABAB receptors (Rs) and acetylcholine (ACh) acting upon muscarinic 2 (AChM2) Rs. Here, we examined whether Mo5 neurons undergo homeostatic regulation through changes in these inhibitory receptors following prolonged activity with enforced waking. By immunofluorescence, we assessed that the proportion of Mo5 neurons positively stained for GABAARs was significantly higher after sleep deprivation (SD, ~65%) than sleep control (SC, ~32%) and that the luminance of the GABAAR fluorescence was significantly higher after SD than SC and sleep recovery (SR). Although, all Mo5 neurons were positively stained for GABABRs and AChM2Rs (100%) in all groups, the luminance of these receptors was significantly higher following SD as compared to SC and SR. We conclude that the density of GABAA, GABAB and AChM2 receptors increases on Mo5 neurons during SD. The increase in these receptors would be associated with increased inhibition in the presence of GABA and ACh and thus a homeostatic down-scaling in the excitability of the Mo5 neurons after prolonged waking and resulting increased susceptibility to muscle hypotonia or atonia along with sleep.
... Additional ACh projections to the XII nucleus originate in the dorsal pontine tegmentum. These projections appear to be relatively scant and primarily originate in the pars-compacta of the PPN and α part of the LDN (448). These cells may have state dependent activity typical of pontine ACh neurons (Fig. 4); some may be maximally active during both wakefulness and REM sleep, others may have relatively selective activity increases during REM sleep, and still others may generate phasic bursts in association with phasic events of REM sleep. ...
Upper airway muscles subserve many essential for survival orofacial behaviors, including their important role as accessory respiratory muscles. In the face of certain predisposition of craniofacial anatomy, both tonic and phasic inspiratory activation of upper airway muscles is necessary to protect the upper airway against collapse. This protective action is adequate during wakefulness, but fails during sleep which results in recurrent episodes of hypopneas and apneas, a condition known as the obstructive sleep apnea syndrome (OSA). Although OSA is almost exclusively a human disorder, animal models help unveil the basic principles governing the impact of sleep on breathing and upper airway muscle activity. This article discusses the neuroanatomy, neurochemistry, and neurophysiology of the different neuronal systems whose activity changes with sleep-wake states, such as the noradrenergic, serotonergic, cholinergic, orexinergic, histaminergic, GABAergic and glycinergic, and their impact on central respiratory neurons and upper airway motoneurons. Observations of the interactions between sleep-wake states and upper airway muscles in healthy humans and OSA patients are related to findings from animal models with normal upper airway, and various animal models of OSA, including the chronic-intermittent hypoxia model. Using a framework of upper airway motoneurons being under concurrent influence of central respiratory, reflex and state-dependent inputs, different neurotransmitters, and neuropeptides are considered as either causing a sleep-dependent withdrawal of excitation from motoneurons or mediating an active, sleep-related inhibition of motoneurons. Information about the neurochemistry of state-dependent control of upper airway muscles accumulated to date reveals fundamental principles and may help understand and treat OSA. © 2016 American Physiological Society. Compr Physiol 6:1801-1850, 2016.
... A neuroprotective action by nAChRs has been proposed against neuroinflammatory processes, glutamate cytotoxicity and, by implication, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and ALS (Dajas-Bailador et al. 2000; Albuquerque et al. 2009; Kawamata et al. 2011; Gao et al. 2014; Egea et al. 2015). To the best of our knowledge, it had not been described before for the hypoglossal nucleus perhaps because cholinergic nerve terminals, from the laterodorsal and pedinculopontine tegmental nuclei, are not abundant and are distributed in an apparently scattered fashion (Rukhadze & Kubin, 2007). A recent study has suggested the presence of motor axon collaterals that may connect motoneurons to their neighbours to increase cholinergic output (Kanjhan et al. 2015). ...
Key points:
Impaired uptake of glutamate builds up the extracellular level of this excitatory transmitter to trigger rhythmic neuronal bursting and delayed cell death in the brainstem motor nucleus hypoglossus. This process is the expression of the excitotoxicity that underlies motoneuron degeneration in diseases such as amyotrophic lateral sclerosis affecting bulbar motoneurons. In a model of motoneuron excitotoxicity produced by pharmacological block of glutamate uptake in vitro, rhythmic bursting is suppressed by activation of neuronal nicotinic receptors with their conventional agonist nicotine. Emergence of bursting is facilitated by nicotinic receptor antagonists. Following excitotoxicity, nicotinic receptor activity decreases mitochondrial energy dysfunction, endoplasmic reticulum stress and production of toxic radicals. Globally, these phenomena synergize to provide motoneuron protection. Nicotinic receptors may represent a novel target to contrast pathological overactivity of brainstem motoneurons and therefore to prevent their metabolic distress and death.
Abstract:
Excitotoxicity is thought to be one of the early processes in the onset of amyotrophic lateral sclerosis (ALS) because high levels of glutamate have been detected in the cerebrospinal fluid of such patients due to dysfunctional uptake of this transmitter that gradually damages brainstem and spinal motoneurons. To explore potential mechanisms to arrest ALS onset, we used an established in vitro model of rat brainstem slice preparation in which excitotoxicity is induced by the glutamate uptake blocker dl-threo-β-benzyloxyaspartate (TBOA). Because certain brain neurons may be neuroprotected via activation of nicotinic acetylcholine receptors (nAChRs) by nicotine, we investigated if nicotine could arrest excitotoxic damage to highly ALS-vulnerable hypoglossal motoneurons (HMs). On 50% of patch-clamped HMs, TBOA induced intense network bursts that were inhibited by 1-10 μm nicotine, whereas nAChR antagonists facilitated burst emergence in non-burster cells. Furthermore, nicotine inhibited excitatory transmission and enhanced synaptic inhibition. Strong neuroprotection by nicotine prevented the HM loss observed after 4 h of TBOA exposure. This neuroprotective action was due to suppression of downstream effectors of neurotoxicity such as increased intracellular levels of reactive oxygen species, impaired energy metabolism and upregulated genes involved in endoplasmic reticulum (ER) stress. In addition, HMs surviving TBOA toxicity often expressed UDP-glucose glycoprotein glucosyltransferase, a key element in repair of misfolded proteins: this phenomenon was absent after nicotine application, indicative of ER stress prevention. Our results suggest nAChRs to be potential targets for inhibiting excitotoxic damage of motoneurons at an early stage of the neurodegenerative process.
... The evidences indicate that cholinergic neurons of the LDT are involved in multitude of functions. For example, cholinergic neurons of the LDT connect with substantia nigra and the ventral tegmental area, influencing dopaminergic cells by M5 receptors and causing release of dopamine in the nucleus accumbens (Segovia et al. 2008; Lammel et al. 2012; Yeomans 2012 ); they connect with the basal forebrain cholinergic neurons (nucleus basalis magnocellularis) that subsequently influence the neocortical arousal (Jones and Cuello 1989; Yeomans 2012 ); they connect with auditory thalamus and cochlear nucleus that were postulated to play a role in novelty detection, sensory gaiting and arousal (Mellott et al. 2011; Schofield et al. 2011); they regulate regional blood flow in some parts of the brain (Koyama et al. 1994 ) as well as connect with the hypoglossal nucleus believed to be involved in control of wakefulness and REM sleep (Rukhadze and Kubin 2007). The AMCS, however, seems to be specifically involved in aversive arousal and initiation of aversive emotional state. ...
The review summarizes evidences from extensive studies suggesting that ascending mesolimbic cholinergic system (AMCS) that terminates in vast areas of forebrain and diencephalic limbic areas is responsible for specific generation of aversive arousal and aversive emotional state. This state is accompanied by emission of threatening and/or alarming vocalizations that served as a quantitative measure of the emotional response. The AMCS originates from the cholinergic neurons within the laterodorsal tegmental nucleus that have widespread and diffuse ascending connections. Activity of the AMCS induced by activation of the muscarinic cholinergic receptors in the terminal fields of this system, or by glutamate stimulation of neurons of the laterodorsal tegmental nucleus, brings about aversive state with alarming vocalizations. It is postulated that release of acetylcholine from the terminals of the AMCS in the vast areas of the forebrain and diencephalon serves as the initiator of the aversive emotional state with concomitant manifestations and alarming vocal signaling. It is concluded that the AMCS serves as a specific physiological, psychological, and social arousing and alarming system.
... The onset of REM sleep is marked by increased activity of cholinergic pontine neurons in the laterodorsal tegmental and pedunculopontine tegmental nuclei (LTD/PPT) [104][105][106] with connections to respiratory neurons in the medulla [107,108]. Injection of cholinergic agonists into the region of the LTD/PPT induces a REM sleep like state with respiratory depression characterized by decreased tidal volumes and respiratory rate [109]. This suggests a connection between the LTD/PPT and the rhythm generating cells in the brainstem. ...
Organophosphate (OP) poisoning is a health issue worldwide with over 200,000 deaths per year. Although not a problem in most developed countries, in some third world countries, one third of a hospital’s population could be patients with OP exposure. Even with the most aggressive therapy, 10-40% of patients admitted to an intensive care unit will die. Research into the best practice for treating OP poisoning is lacking, due somewhat to a lack of detailed understanding of the physiology of OP poisoning. Our research uses animal models of acute OP poisoning to explore the mechanism of OP-induced respiratory failure.
Our research shows that animals poisoned with dichlorvos demonstrated a uniformly fatal central apnea that, if prevented, was followed immediately by a variable pulmonary dysfunction. Potential mechanisms for dichlorvos-induced central apnea can be divided into direct effects on the central respiratory oscillator (CRO) and feedback inhibition of the CRO. Two afferent pathways that can induce apnea include vagal feedback pathways and feed-forward pathways from the cerebral hemispheres. In our studies we found that vagal feedback and feed forward inhibition from the cerebral hemispheres were not required for OP-induced central apnea. The pre-Botzinger complex in the brainstem is thought to be the kernel of the CRO, but exposure of the pre-Botzinger complex to dichlorvos was not sufficient for apnea. Although OP induced central apnea was uniformly fatal, partial recovery of the CRO occurred post apnea with mechanical ventilation.
Central apnea was ubiquitous in our rat poisoning model, but pulmonary dysfunction was extremely variable, with a range of pulmonary effects from fulminate pulmonary failure with prominent pulmonary secretions to no pulmonary dysfunction at all. Vagal efferent activity is involved in neural control of pulmonary tissue but the vagus was not involved in OP-induced pulmonary dysfunction. Anti-muscarinic medications are the mainstay of clinical therapy and are commonly dosed by their effects on pulmonary secretions. Our studies found that atropine (the most common therapeutic agent for OP poisoning) resulted in a ventilation-perfusion mismatch secondary to effects on the pulmonary vasculature.
... This caudal pole of the PPTn is neurophysiologically distinct, since unlike more rostral PPTn locations, neurons in this caudal region are REM sleep-active, project to the PRF (Jones, 1990; Semba et al., 1990; Semba and Fibiger, 1992; Kohlmeier et al., 2002) and thalamus (Semba et al., 1990; Semba and Fibiger, 1992; Bevan and Bolam, 1995), and are thought to modulate REM sleep and the associated electrocortical activation (Steriade and McCarley, 2005a,b). Neurons in the caudal pole of the PPTn also project to the areas of the rostral ventrolateral medulla (Yasui et al., 1990 ) that contain respiratory neurons critical to the generation of respiratory rhythm and pattern (Feldman and Del Negro, 2006), and to motor pools such as the hypoglossal motor nucleus which innervates the genioglossus muscle of the tongue (Woolf and Butcher, 1989; Fay and Norgren, 1997; Rukhadze and Kubin, 2007). At the end of surgery, all the electrodes were connected to pins and inserted into a miniature plug (STC-89PI-220ABS, Carleton University, Ottawa, ON, Canada). ...
Serotonin type 1A (5-HT(1A)) receptor-responsive neurons in the pedunculopontine tegmental nucleus (PPTn) become maximally active immediately before and during rapid eye movement (REM) sleep. A prevailing model of REM sleep generation indicates that activation of such neurons contributes significantly to the generation of REM sleep, and if correct then inactivation of such neurons ought to suppress REM sleep. We test this hypothesis using bilateral microperfusion of the 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 10 μm) into the PPTn; this tool has been shown to selectively silence REM sleep-active PPTn neurons while the activity of wake/REM sleep-active PPTn neurons is unaffected. Contrary to the prevailing model, bilateral microperfusion of 8-OH-DPAT into the PPTn (n = 23 rats) significantly increased REM sleep both as a percentage of the total recording time and sleep time, compared with both within-animal vehicle controls and between-animal time-controls. This increased REM sleep resulted from an increased frequency of REM sleep bouts but not their duration, indicating an effect on mechanisms of REM sleep initiation but not maintenance. Furthermore, an increased proportion of the REM sleep bouts stemmed from periods of low REM sleep drive quantified electrographically. Targeted suppression of 5-HT(1A) receptor-responsive PPTn neurons also increased respiratory rate and respiratory-related genioglossus activity, and increased the frequency and amplitude of the sporadic genioglossus activations occurring during REM sleep. These data indicate that 5-HT(1A) receptor-responsive PPTn neurons normally function to restrain REM sleep by elevating the drive threshold for REM sleep induction, and restrain the expression of respiratory rate and motor activities.
... The present study also showed that neurochemical excitation at certain PPT injection sites evoked an activation of genioglossus EMG. Consistent with this observation, Rukhadze and Kubin (2007) have shown that cholinergic neurons of the PPT, originating in a discrete portion of its pars compacta, send bilateral projections to the motor nucleus of the hypoglossal nerve (Mo12). Thus, it is possible, that these projections contribute to both excitatory and inhibitory modulation of the activity of hypoglossal motoneurons. ...
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.
... Descending projections originating from mesopontine cholinergic neurons generally play a role in decreasing muscle tone during rapid eye movement (REM) sleep [144]. As an exception, these neurons have been shown to increase activity in hypoglossal motor neurons during waking and REM sleep [129]. ...
... If brainstem premotor noradrenergic and serotonergic neurons mediate only a small fraction of motoneuronal activation from the PF region of the posterior hypothalamus, one needs to consider other pathways. Those that may be proposed based on the existing neuroanatomical and pharmacological data include direct excitatory projections of ORX neurons to motoneurons [13,22,33], ORX-mediated activation of hypothalamic histaminergic neurons that may, in turn, activate motoneurons [24,34], brainstem cholinergic neurons [20,28], or other non-adrenergic/ non-serotonergic premotor neurons that are excited by pathways that descend from the posterior, lateral hypothalamus [6]. Since PF bicuculline also excites many non-ORX cells in the PF region [21], such neurons may mediate motoneuronal activation from the posterior hypothalamus through pathways that use transmitters other than those classically considered important for wake-related motor activation. ...
The perifornical (PF) region of the posterior hypothalamus plays an important role in the regulation of sleep-wake states and motor activity. Disinhibition of PF neurons by the GABA(A) receptor antagonist, bicuculline, has been used to study the mechanisms of wake- and motor activity-promoting effects that emanate from the PF region. Bicuculline activates PF neurons, including the orexin-containing cells that have major excitatory projections to brainstem noradrenergic and serotonergic neurons. Since premotor aminergic neurons are an important source of motoneuronal activation, we hypothesized that they mediate the excitation of motoneurons that results from disinhibition of PF neurons with bicuculline. In urethane-anesthetized, paralyzed and artificially ventilated rats, we found that PF bicuculline injections (1mM, 20 nl) made after combined microinjections into the hypoglossal (XII) nucleus of alpha(1)-adrenergic and serotonergic receptor antagonists (prazosin and methysergide) increased XII nerve activity by 80+/-16% (SE) of the control activity level. Thus, activation of XII motoneurons originating in the hypothalamic PF region was not abolished despite effective elimination by the aminergic antagonists of the endogenous noradrenergic and serotonergic excitatory drives to XII motoneurons and abolition of XII motoneuronal activation by exogenous serotonin or phenylephrine. These results show that a major component of XII motoneuronal activation originating in the posterior hypothalamus is mediated by pathways other than the noradrenergic and serotonergic projections to motoneurons.
... Cholinergic neurons in the PPT/LDT have descending projections to the medullary reticular nuclei and the lateral reticular nucleus [40,41] , probably including the preBötC. Cholinergic neurons in the PPT/LDT also project to motor nuclei of cranial nerves including XII nucleus [40][41][42] . Numerous cholinergic neurons are found in the medullary reticular formation and near the ventral medullary surface [43][44][45] . ...
Nicotinic acetylcholine receptors (nAChRs) are expressed in brainstem and spinal cord regions involved in the control of breathing. These receptors mediate central cholinergic regulation of respiration and effects of the exogenous ligand nicotine on respiratory pattern. Activation of alpha4* nAChRs in the preBötzinger Complex (preBötC), an essential site for normal respiratory rhythm generation in mammals, modulates excitatory glutamatergic neurotransmission and depolarizes preBötC inspiratory neurons, leading to increases in respiratory frequency. nAChRs are also present in motor nuclei innervating respiratory muscles. Activation of post- and/or extra-synaptic alpha4* nAChRs on hypoglossal (XII) motoneurons depolarizes these neurons, potentiating tonic and respiratory-related rhythmic activity. As perinatal nicotine exposure may contribute to the pathogenesis of sudden infant death syndrome (SIDS), we discuss the effects of perinatal nicotine exposure on development of the cholinergic and other neurotransmitter systems involved in control of breathing. Advances in understanding of the mechanisms underlying central cholinergic/nicotinic modulation of respiration provide a pharmacological basis for exploiting nAChRs as therapeutic targets for neurological disorders related to neural control of breathing such as sleep apnea and SIDS.
... The expression of nAChRs in cardiovascular, respiratory, and motor nuclei in the human fetal and infant medulla is supported by extensive animal data (Swanson et al., 1987;Hellstrom-Lindahl et al., 1998;Ferreira et al., 2000;Cucchiaro and Commons, 2003;Kubin and Fenik, 2004;Padley et al., 2007). Rodent studies suggest that the cholinergic projections to medullary nuclei are primarily from the pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus in the mesopons that are critical for arousal and the generation of REM sleep, suggesting that nAChRs also play an important role in these components of state regulation (Frank et al., 2001;Lena at al., 2004;Padley et al., 2007;Rukhadze and Kubin, 2007). Of note, prenatal nicotine exposure interrupts normal sleep/wake cycling by reducing periods of REM sleep in the neonatal rat (Frank et al., 2001), and β2 knockout mice experience increased periods of REM (Lena et al., 2004) and impaired respiration which is enhanced upon nicotine exposure (Cohen et al., 2002). ...
Maternal cigarette smoking during pregnancy adversely affects fetal development and increases the risk for the sudden infant death syndrome (SIDS). In SIDS we have reported abnormalities in the medullary serotonergic (5-HT) system, which is vital for homeostatic control. In this study we analyzed the inter-relationship between nicotinic receptors (nAChRs), to which nicotine in cigarette smoke bind, and the medullary 5-HT system in the human fetus and infant as a step towards determining the mechanisms whereby smoking increases SIDS risk in infants with 5-HT defects. Immunohistochemistry for the alpha4 nAChR subunit and 5-HT neurons was applied in fetal and infant medullae (15-92 postconceptional weeks, n=9). The distribution of different nAChRs was determined from 39-82 postconceptional weeks (n=5) using tissue autoradiography for 3H-nicotine, 3H-epibatidine, 3H-cytisine, and 125I-bungarotoxin; the findings were compared to laboratory 5-HT1A and 5-HT transporter binding data, and 5-HT neuronal density. Alpha4 immunoreactivity was ubiquitously expressed in medullary nuclei related to homeostatic functions from 15 weeks on, including rhombic lip germinal cells. At all ages, alpha4 co-localized with 5-HT neurons, indicating a potential site of interaction whereby exogenous nicotine may adversely affect 5-HT neuronal development and function. Binding for heteromeric nAChRs was highest in the inferior olive, and for homomeric nAChRs, in the vagal complex. In the paragigantocellularis lateralis, 5-HT1A receptor binding simultaneously increased as alpha7 binding decreased across infancy. This study indicates parallel dynamic and complex changes in the medullary nicotinic and 5-HT systems throughout early life, i.e., the period of risk for SIDS.
... The identification of a secondary state-dependent suppression component to this reflex arc has raised the possibility that more pronounced reflex inhibition rather than a loss of excitation may mediate diminished pharyngeal reflex responses during sleep (26). Indeed, advances in our understanding of the neuroanatomy of the genioglossus negative pressure reflex and hypoglossal motor nucleus inputs from rat studies have highlighted the extensive presence of inhibitory inputs to the genioglossus muscle (28)(29)(30). Nonetheless, although genioglossus muscle responsiveness may be impaired during sleep compared with wakefulness, it is clear that the muscle does respond to sustained negative pressure ( Figure 1) and potentially hypercapnia, particularly when combinations of stimuli are provided (31)(32)(33). ...
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.
... Using avidin–biotin–horseradish peroxidase histochemistry (Vector, Burlinghame, CA, USA), with and then without heavy metal intensification, c-fos expressed in neuronal nuclei was stained black, and prepro-ORX-expressing cells, brown. The same approach was applied to brainstem sections to visualize c-fos in pontomedullary catecholaminergic neurones labelled for tyrosine hydroxylase (1 : 35 000; Sigma; Rukhadze & Kubin, 2007a) and pontine cholinergic neurones immunohistochemically labelled for nitric oxide synthase (1 : 5000; Sigma) (Vincent & Kimura, 1992; Rukhadze & Kubin, 2007b). ...
Studies in behaving animals suggest that neurones located in the perifornical (PF) region of the posterior hypothalamus promote wakefulness and suppress sleep. Among such cells are those that synthesize the excitatory peptides, orexins (ORX). Lack of ORX, or their receptors, is associated with narcolepsy/cataplexy, a disorder characterized by an increased pressure for rapid eye movement (REM) sleep. We used anaesthetized rats in which pontine microinjections of a cholinergic agonist, carbachol, can repeatedly elicit REM sleep-like episodes to test whether activation of PF cells induced by antagonism of endogenous, GABA(A) receptor-mediated, inhibition suppresses the ability of the brainstem to generate REM sleep-like state. Microinjections of the GABA(A) receptor antagonist, bicuculline (20 nl, 1 mm), into the PF region elicited cortical and hippocampal activation, increased the respiratory rate and hypoglossal nerve activity, induced c-fos expression in ORX and other PF neurones, and increased c-fos expression in pontine A7 and other noradrenergic neurones. The ability of pontine carbachol to elicit any cortical, hippocampal or brainstem component of the REM sleep-like response was abolished during the period of bicuculline-induced activation. The activating and REM sleep-suppressing effect of PF bicuculline was not attenuated by systemic administration of the ORX type 1 receptor antagonist, SB334867. Thus, activation of PF neurones that are endogenously inhibited by GABA(A) receptors is sufficient to turn off the brainstem REM sleep-generating network; the effect is, at least in part, due to activation of pontine noradrenergic neurones, but is not mediated by ORX type 1 receptors. A malfunction of the pathway that originates in GABA(A) receptor-expressing PF neurones may cause narcolepsy/cataplexy.
... The identification of a secondary state-dependent suppression component to this reflex arc has raised the possibility that more pronounced reflex inhibition rather than a loss of excitation may mediate diminished pharyngeal reflex responses during sleep (26). Indeed, advances in our understanding of the neuroanatomy of the genioglossus negative pressure reflex and hypoglossal motor nucleus inputs from rat studies have highlighted the extensive presence of inhibitory inputs to the genioglossus muscle (28)(29)(30). Nonetheless, although genioglossus muscle responsiveness may be impaired during sleep compared with wakefulness, it is clear that the muscle does respond to sustained negative pressure ( Figure 1) and potentially hypercapnia, particularly when combinations of stimuli are provided (31)(32)(33). ...
Obstructive sleep apnea (OSA) is a common disorder characterized by repetitive narrowing or collapse of the pharyngeal airway during sleep. The disorder is associated with major comorbidities including excessive daytime sleepiness and increased risk of cardiovascular disease. The underlying pathophysiology is multifactorial and may vary considerably between individuals. Important risk factors include obesity, male sex, and aging. However, the physiological mechanisms underlying these risk factors are not clearly understood. This brief review summarizes the current understanding of OSA pathophysiology in adults and highlights the potential mechanisms underlying the principal risk factors. In addition, some of the pathophysiological characteristics associated with OSA that may modulate disease severity are illustrated. Finally, the potential for novel treatment strategies, based on an improved understanding of the underlying pathophysiology, is also discussed with the ultimate aim of stimulating research ideas in areas where knowledge is lacking.
The chapter provides an introduction to the mechanism underlying the generation and regulation of sleep under physiologic conditions. Sleep–wake behavior is gated by circadian rhythm and paced by the ultradian rhythm called the basic rest–activity cycle (BRAC). Within the framework of these two rhythms, three distinct behavioral states—wakefulness, non-rapid eye movement (NREM) sleep, and REM sleep—are generated by neuronal groups that differ based on their neurotransmitter phenotypes, anatomic locations, and relationship of their activity to the phases of the sleep–wake cycle. Gamma-aminobutyric acid (GABA) plays a major role in this network, with different groups of GABAergic cells contributing to the generation of each of the three behavioral states. Different groups of cholinergic cells support wakefulness or REM sleep. Norepinephrine-, serotonin-, histamine-, and orexin-containing neurons subserve different aspects of wakefulness. The entire network possesses multiple mechanisms that support the homeostatic regulation of sleep. These include the use-dependent control of neurotransmitter synthesis, neurotransmitter receptor trafficking, cellular effects of metabolites (e.g., adenosine), response to depletion of energy stores (e.g., glycogen), as well as actions of sleep-promoting cytokines and growth factors responsive to inflammation and external environment (e.g., synaptic plasticity supporting memory).KeywordsAdenosineCircadian rhythmGABASleep homeostasisHypothalamusPonsUltradian rhythm
In obstructive sleep apnea (OSA) patients, contraction of the muscles of the tongue is needed to protect the upper airway from collapse. During wakefulness, norepinephrine directly excites motoneurons that innervate the tongue and other upper airway muscles but its excitatory effects decline during sleep, thus contributing to OSA. In addition to motoneurons, NE may regulate activity in premotor pathways but little is known about these upstream effects. To start filling this void, we injected a retrograde tracer (beta-subunit of cholera toxin-CTb; 5–10 nl, 1%) into the hypoglossal (XII) motor nucleus in 7 rats. We then used dual immunohistochemistry and brightfield microscopy to count dopamine beta-hydroxylase (DBH)-positive axon terminals closely apposed to CTb cells located in five anatomically distinct XII premotor regions. In different premotor groups, we found on the average 2.2–4.3 closely apposed DBH terminals per cell, with ˜60% more terminals on XII premotor neurons located in the ventrolateral pontine parabrachial region and ventral medullary gigantocellular region than on XII premotor cells of the rostral or caudal intermediate medullary reticular regions. This difference suggests stronger control by norepinephrine of the interneurons that mediate complex behavioral effects than of those mediating reflexes or respiratory drive to XII motoneurons.
The hypoglossal nucleus, the nucleus of the twelfth cranial nerve, is located dorsally in the midline of the medulla oblongata. The hypoglossal nucleus contains lower motor neurons which innervate the tongue muscles that control tongue movements involved in speech production, swallowing, mastication and associated respiratory movements. GABA A and glycine receptors are heteropentameric ionotropic receptors that facilitate fast-response, inhibitory neurotransmission in the mammalian brain and spinal cord. We investigated the immunohistochemical distribution of the GABA A receptor α 1 , α 2 , β 2,3 subunits and glycine receptors as well as their relationship to the vesicular GABA transporter (VGAT) in the human hypoglossal nucleus at the light and confocal laser scanning microscope levels. The results showed that all of the GABA A receptor subunits as well as glycine receptor display punctate labelling indicative of synapses on the soma and dendritic membranes of large neurons within the hypoglossal nucleus. On average, approximately 50% of glycine receptors were co localised with GABA A receptor α 1 subunits. Also on average GABA A α 2 and β 2,3 subunits were colocalised with approximately 30% of glycine receptor subunits. VGAT positive terminals were associated with both GABA A and glycine receptor types. Both glycinergic and GABAergic positive puncta were found adjacent to VGAT terminal-like staining. These results suggest that inhibition of human hypoglossal motor neurons occurs not only through complex interaction of separated GABA A R and glycine receptor regions, but also through synapses containing both inhibitory receptor types co-existing at the same synaptic sites.
В статье приводятся данные макро, и микроскопического исследования продолговатого мозга дельфинов вида phocoena phocoena relicta. Акцентируется внимание на различной информационной значимости гистологических и МРТ исследований ткани головного мозга. Определены наиболее выраженные структуры анатомии продолговатого мозга, и их связь с цитоархитектоникой и гистологией соматосенсорных, соматомоторных ядерных образований и ассоциативных формаций этого отдела центральной нервной системы. В статье описана поуровневая гистотопографическая характеристика medulla oblongata, и предпринята попытка оценки степени развития отдельных ядер в связи с особенностями физиологии Phocoena phocoena relicta.
Muscles of the face, tongue and jaws are innervated by motoneurons in the lower brainstem. In most instances, the locations of motoneurons innervating specific muscles have been well characterized. Most of the projections to the oromotor nuclei originate from the brainstem, although in a few instances, cortical and hypothalamic projections have also been identified. Brainstem pathways include projections from orosensory structures (e.g., the trigeminal sensory complex), the nucleus of the solitary tract and the parabrachial nuclei, as well as noradrenergic and serotonergic input from raphe and catecholaminergic nuclei. In addition, midbrain projections to specific subdivisions of the facial nucleus mediate vibrissae, orbital and pinna movements. A large proportion of the projections to the oromotor nuclei, however are from specific regions of the brainstem reticular formation, within which are neurons with projections to multiple oromotor nuclei. These reticular formation structures are integral to the ororhythmic responses of mastication, licking, swallowing, respiration, and whisking.
NAP (davunetide) is a novel neuroprotective compound with mechanism of action that appears to involve microtubule (MT) stabilization and repair. To evaluate, for the first time, the impact of NAP on axonal transport in vivo and to translate it to neuroprotection in a severe neurodegeneration, the SOD1-G93A mouse model for amyotrophic lateral sclerosis (ALS) was used. Manganese-enhanced magnetic resonance imaging (MRI), estimating axonal transport rates, revealed a significant reduction of the anterograde axonal transport in the ALS mice compared to healthy control mice. Acute NAP treatment normalized axonal transport rates in these ALS mice. Tau hyperphosphorylation, associated with MT dysfunction and defective axonal transport, was discovered in the brains of the ALS mice and was significantly reduced by chronic NAP treatment. Furthermore, in healthy wild type (WT) mice, NAP reversed axonal transport disruption by colchicine, suggesting drug-dependent protection against axonal transport impairment through stabilization of the neuronal MT network. Histochemical analysis showed that chronic NAP treatment significantly protected spinal cord motor neurons against ALS-like pathology. Sequential MRI measurements, correlating brain structure with ALS disease progression, revealed a significant damage to the ventral tegmental area (VTA), indicative of impairments to the dopaminergic pathways relative to healthy controls. Chronic daily NAP treatment of the SOD1-G93A mice, initiated close to disease onset, delayed degeneration of the trigeminal, facial and hypoglossal motor nuclei as was significantly apparent at day 90-100 and further protected the VTA throughout life. Importantly, protection of the VTA was significantly correlated with longevity and overall, NAP treatment significantly prolonged life span in the ALS mice.
Rationale:
Inhibition of pharyngeal motoneurons accompanies REM sleep and is a cause of hypoventilation and obstructive sleep apnea in humans. One explanation posits that the neurotransmitters glycine and γ-aminobutyric acid are responsible for REM sleep motor inhibition. However, blockade of that mechanism at cranial motor nuclei increases motor activity in all sleep-wake states, and least of all in REM sleep, arguing against it as a major mechanism of REM sleep pharyngeal motor inhibition.
Objectives:
To identify the mechanism of REM sleep inhibition at the hypoglossal motor pool.
Methods:
Genioglossus and diaphragm activities were recorded in 34 rats across sleep-wake states. Microdialysis probes were implanted into the hypoglossal motor pool.
Measurements and main results:
Here we show that muscarinic receptor antagonism at the hypoglossal motor pool prevents the inhibition of genioglossus activity throughout REM sleep; likewise, with G-protein-coupled inwardly rectifying potassium (GIRK) channel blockade. Importantly, the genioglossus activating effects of these interventions were largest in REM sleep and minimal or often absent in other sleep-wake states. Finally, we showed that muscarinic inhibition of the genioglossus is functionally linked to GIRK channel activation.
Conclusions:
We identify a powerful cholinergic-GIRK channel mechanism operating at the hypoglossal motor pool that has its largest inhibitory influence in REM sleep and minimal or no effects in other sleep-wake states. This mechanism is the major cause of REM sleep inhibition at a pharyngeal motor pool critical for effective breathing.
In many brain areas, few cholinergic synapses are identified. Acetylcholine is released into the extracellular space and acts through diffuse transmission. Motoneurons, however, are contacted by numerous cholinergic terminals, indicating synaptic cholinergic transmission on them. The muscarinic m2 receptor is the major acetylcholine receptor subtype of motoneurons, therefore, we analyzed the localization of m2 receptor in correlation with synapses by electron microscopic immunohistochemistry in the mouse trigeminal, facial and hypoglossal motor nuclei. In all nuclei, m2 receptors were localized at the membrane of motoneuronal perikarya and dendrites. The m2 receptors were concentrated at cholinergic synapses located on the perikarya and most proximal dendrites. However, m2 receptors at cholinergic synapses represented only a minority (<10%) of surface m2 receptors. The m2 receptors were also enriched at glutamatergic synapses both in motoneuronal perikarya and dendrites. A relatively large proportion (20-30%) of plasma membrane-associated m2 receptors were located at glutamatergic synapses. In conclusion, the effect of acetylcholine on motoneuron populations might be mediated through synaptic as well as diffuse type of transmission. J. Comp. Neurol., 2012. © 2012 Wiley Periodicals, Inc.
In brain stem slices from neonatal (postnatal days 0-4) CD-1 mice, muscarinic ACh receptors (MAChRs) increased rhythmic inspiratory-related and tonic hypoglossal nerve discharge and depolarized single hypoglossal motoneurons (HMs) via an inward current without changing input resistance. These responses were blocked by the MAChR antagonist 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; 100 nM). MAChRs shifted voltage-dependent activation of the hyperpolarization-activated cation current to more positive levels. MAChRs increased the HM repetitive firing rate and decreased rheobase, with both effects being blocked by 4-DAMP. Muscarinic agonists reduced the afterhyperpolarization of single action potentials (APs), suggesting that small-conductance Ca(2+)-dependent K(+) current inhibition increased the HM firing rate. Muscarinic agonists also reduced the AP amplitude and slowed its time course, suggesting that MAChRs inhibited voltage-gated Na(+) channels. To compare muscarinic excitation of single HMs to muscarinic excitatory effects on motor output in thicker brain stem slices requiring higher extracellular K(+) for rhythmic activity, we tested the effects of muscarinic agonists on single HM excitability in high-K(+) artificial cerebrospinal fluid (aCSF). In high-K(+) aCSF, muscarinic agonists still depolarized HMs and altered AP size and shape, as in standard aCSF, but did not increase the steady-state firing rate, decrease afterhyperpolarization, or alter threshold potential. These results indicate that the basic cellular response of HMs to muscarinic receptors is excitatory, via a number of distinct mechanisms, and that this excitatory response will be largely preserved in rhythmically active brain stem slices.
The superior salivatory nucleus (SSN) contains preganglionic parasympathetic neurons to the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor agonist, stimulates the salivary glands and is presently used as sialogogue in the treatment of dry mouth. Since cevimeline passes through the blood-brain barrier, it is also able to act on muscarinic acetylcholine receptors in the central nervous system. Our preliminary experiment using the whole-cell patch-clamp technique has shown that cevimeline excites SSN neurons in rat brain slices, suggesting that SSN neurons have muscarinic acetylcholine receptors; however, it is unclear which subtypes of muscarinic acetylcholine receptors exist in SSN neurons. In the present study, we investigated immunohistochemically muscarinic acetylcholine receptor subtypes, M1 receptor (M1R), M2R, M3R, M4R, and M5R in SSN neurons. SSN neurons innervating the salivary glands, retrogradely labeled with a fluorescent tracer from the chorda-lingual nerve, mostly expressed M3R immunoreactivity (-ir) (92.3%) but not M1R-ir. About half of such SSN neurons also showed M2R- (40.1%), M4R- (54.0%) and M5R-ir (46.0%); therefore, it is probable that SSN neurons co-express M3R-ir with at least two of the other muscarinic receptor subtypes. This is the first report to show that SSN neurons contain muscarinic acetylcholine receptors.
The intrinsic laryngeal muscles are differentially modulated during respiration as well as other states and behaviors such as hypocapnia and sleep. Previous anatomical and pharmacological studies indicate a role for acetylcholine at the level of the nucleus ambiguus in the modulation of laryngeal motoneuron (LMN) activity. The present study investigated the anatomical nature of cholinergic input to inspiratory- (ILM) and expiratory-modulated (ELM) laryngeal motoneurons in the loose formation of the nucleus ambiguus. Using combined in vivo intracellular recording, dye filling, and immunohistochemistry, we demonstrate that LMNs identified in Sprague-Dawley rat receive several close appositions from vesicular acetylcholine transporter-immunoreactive (VAChT-ir) boutons. ELMs receive a significantly greater number of close appositions (mean ± standard deviation [SD]: 47 ± 11; n = 5) than ILMs (32 ± 9; n = 8; t-test P < 0.05). For both LMN types, more close appositions were observed on the cell soma and proximal dendrites compared to distal dendrites (two-way analysis of variance [ANOVA], P < 0.0001). Using fluorescence confocal microscopy, almost 90% of VAChT-ir close appositions (n = 45 boutons on n = 4 ELMs) were colocalized with the synaptic marker synaptophysin. These results support a strong influence of cholinergic input on LMNs and may have implications in the differential modulation of laryngeal muscle activity.
Sleep-induced apnea and disordered breathing refers to intermittent, cyclical cessations or reductions of airflow, with or without obstructions of the upper airway (OSA). In the presence of an anatomically compromised, collapsible airway, the sleep-induced loss of compensatory tonic input to the upper airway dilator muscle motor neurons leads to collapse of the pharyngeal airway. In turn, the ability of the sleeping subject to compensate for this airway obstruction will determine the degree of cycling of these events. Several of the classic neurotransmitters and a growing list of neuromodulators have now been identified that contribute to neurochemical regulation of pharyngeal motor neuron activity and airway patency. Limited progress has been made in developing pharmacotherapies with acceptable specificity for the treatment of sleep-induced airway obstruction. We review three types of major long-term sequelae to severe OSA that have been assessed in humans through use of continuous positive airway pressure (CPAP) treatment and in animal models via long-term intermittent hypoxemia (IH): 1) cardiovascular. The evidence is strongest to support daytime systemic hypertension as a consequence of severe OSA, with less conclusive effects on pulmonary hypertension, stroke, coronary artery disease, and cardiac arrhythmias. The underlying mechanisms mediating hypertension include enhanced chemoreceptor sensitivity causing excessive daytime sympathetic vasoconstrictor activity, combined with overproduction of superoxide ion and inflammatory effects on resistance vessels. 2) Insulin sensitivity and homeostasis of glucose regulation are negatively impacted by both intermittent hypoxemia and sleep disruption, but whether these influences of OSA are sufficient, independent of obesity, to contribute significantly to the "metabolic syndrome" remains unsettled. 3) Neurocognitive effects include daytime sleepiness and impaired memory and concentration. These effects reflect hypoxic-induced "neural injury." We discuss future research into understanding the pathophysiology of sleep apnea as a basis for uncovering newer forms of treatment of both the ventilatory disorder and its multiple sequelae.
Rapid diagnosis of volume overload in patients with suspected congestive heart failure (CHF) is necessary for the timely administration of therapeutic agents. We sought to use the measurement of respiratory variation of inferior vena cava (IVC) diameter as a diagnostic tool for identification of CHF in patients presenting with acute dyspnea.
The IVC was measured sonographically during a complete respiratory cycle of 46 patients meeting study criteria. Percentage of respiratory variation of IVC diameter was compared to the diagnosis of CHF or alternative diagnosis.
Respiratory variation of IVC was smaller in patients with CHF (9.6%) than without CHF (46%) and showed good diagnostic accuracy with area under the receiver operating characteristic curve of 0.96. Receiver operating characteristic curve analysis showed optimum cutoff of 15% variation or less of IVC diameter with 92% sensitivity and 84% specificity for the diagnosis of CHF.
Inferior vena cava ultrasound is a rapid, reliable means for identification of CHF in the acutely dyspneic patient.
The inspiratory drive to hypoglossal (XII) motoneurons originates in the caudal medullary intermediate reticular (IRt) region. This drive is mainly glutamatergic, but little is known about the neurochemical features of IRt XII premotor neurons. Prompted by the evidence that XII motoneuronal activity is controlled by both muscarinic (M) and nicotinic cholinergic inputs and that the IRt region contains cells that express choline acetyltransferase (ChAT), a marker of cholinergic neurons, we investigated whether some IRt XII premotor neurons are cholinergic. In seven rats, we applied single-cell reverse transcription-polymerase chain reaction to acutely dissociated IRt neurons retrogradely labeled from the XII nucleus. We found that over half (21/37) of such neurons expressed mRNA for ChAT and one-third (13/37) also had M2 receptor mRNA. In contrast, among the IRt neurons not retrogradely labeled, only 4 of 29 expressed ChAT mRNA (P < 0.0008) and only 3 of 29 expressed M2 receptor mRNA (P < 0.04). The distributions of other cholinergic receptor mRNAs (M1, M3, M4, M5, and nicotinic alpha4-subunit) did not differ between IRt XII premotor neurons and unlabeled IRt neurons. In an additional three rats with retrograde tracers injected into the XII nucleus and ChAT immunohistochemistry, 5-11% of IRt XII premotor neurons located at, and caudal to, the area postrema were ChAT positive, and 27-48% of ChAT-positive caudal IRt neurons were retrogradely labeled from the XII nucleus. Thus the pre- and postsynaptic cholinergic effects previously described in XII motoneurons may originate, at least in part, in medullary IRt neurons.
Hypoglossal motoneurons are influenced by a variety of neuromodulators, some of which change dynamically across sleep-wake states to alter motoneuron excitability and responses to pharmacological manipulations. Determining the mechanisms underlying the modulation of hypoglossal motoneurons during sleep is relevant to understanding the increased upper airway resistance, airflow limitation and hypoventilation in normal sleeping individuals, as well as the airway obstruction underlying the pathogenesis of obstructive sleep apnea. This review summarizes current concepts underlying the neurobiology of sleep and arousal states, and so identifies the rationale for focus on particular neuromodulators and their effects on hypoglossal motoneurons. Emphasis is placed on the control of hypoglossal motoneurons by components of the aminergic arousal system, i.e., serotonergic, noradrenergic and histaminergic inputs. The role of inhibitory mechanisms at the hypoglossal motor nucleus, including glycine and gamma-amino butyric acid, and the mixed excitatory and inhibitory influences of acetylcholine are also reviewed. New concepts on the control of hypoglossal motoneurons by sedative hypnotics are also discussed.
The present study was undertaken to examine the cholinergic innervation of the brainstem reticular formation in an effort to understand the potential role of cholinergic neurons in processes of sensory-motor modulation and state control. The cholinergic cells and processes within the pontomedullary reticular formation were studied in the rat by application of peroxidase-antiperoxidase immunohistochemistry with silver intensification for cholineacetyltransferase (ChAT). ChAT-immunoreactive cells were located in the pontomesencephalic tegmentum within the laterodorsal and pedunculopontine tegmental (LDT and PPT) nuclei, where they numbered approximately 3,000 on each side and were scattered in the midline, medial, and lateral medullary reticular formation, where they numbered approximately 10,000 in total on each side. The cholinergic neurons within the reticular formation were commonly medium in size and gave rise to multiple dendrites that extended for considerable distances within the periventricular gray or the reticular formation, as is typical of other isodendritic reticular neurons. A prominent innervation of the entire pontomedullary reticular formation was evident by varicose ChAT-immunoreactive fibers that often surrounded large noncholinergic reticular neurons in a typical perisomatic pattern of termination, suggesting a potent influence of the cholinergic innervation on pontomedullary reticular neurons.
This study was performed to examine the hypothesis that thalamic-projecting neurons of mesopontine cholinergic nuclei display activity patterns that are compatible with their role in inducing and maintaining activation processes in thalamocortical systems during the states of waking (W) and rapid-eye-movement (REM) sleep associated with desynchronization of the electroencephalogram (EEG). A sample of 780 neurons located in the peribrachial (PB) area of the pedunculopontine tegmental nucleus and in the laterodorsal tegmental (LDT) nucleus were recorded extracellularly in unanesthetized, chronically implanted cats. Of those neurons, 82 were antidromically invaded from medial, intralaminar, and lateral thalamic nuclei: 570 were orthodromically driven at short latencies from various thalamic sites: and 45 of the latter elements are also part of the 82 cell group, as they were activated both antidromically and synaptically from the thalamus. There were no statistically significant differences between firing rates in the PB and LDT neuronal samples. Rate analyses in 2 distinct groups of PB/LDT neurons, with fast (greater than 10 Hz) and slow (less than 2 Hz) discharge rates in W, indicated that (1) the fast-discharging cell group had higher firing rates in W and REM sleep compared to EEG-synchronized sleep (S), the differences between all states being significant (p less than 0.0005); (2) the slow-discharging cell group increased firing rates from W to S and further to REM sleep, with significant difference between W and S (p less than 0.01), as well as between W or S and REM sleep (p less than 0.0005). Interspike interval histograms of PB and LDT neurons showed that 75% of them have tonic firing patterns, with virtually no high-frequency spike bursts in any state of the wake-sleep cycle. We found 22 PB cells that discharged rhythmic spike trains with recurring periods of 0.8-1 sec. Autocorrelograms revealed that this oscillatory behavior disappeared when their firing rate increased during REM sleep. Dynamic analyses of sequential firing rates throughout the waking-sleep cycle showed that none of the full-blown states of vigilance is associated with a uniform level of spontaneous firing rate. Signs of decreased discharge frequencies of mesopontine neurons appeared toward the end of quiet W, preceding by about 10-20 sec the most precocious signs of EEG synchronization heralding the sleep onset. During transition from S to W, rates of spontaneous discharges increased 20 sec before the onset of EEG desynchronization.(ABSTRACT TRUNCATED AT 400 WORDS)
The neural control of the accessory respiratory muscles regulating upper airway patency is poorly understood. This is particularly true with regard to the declines in electromyographic (EMG) activity of upper airway muscles during sleep. To specify the cellular mechanisms causing decreased upper airway muscle tone during sleep, we used an established pharmacological model of rapid eye movement (REM) sleep. With this model, a REM sleep-like state was reliably produced by microinjecting the cholinergic agonist carbachol directly into the pontine reticular formation of the cat. EMG recording were taken from the posterior cricoarytenoid (PCA) muscles of the larynx during wakefulness and the carbachol-induced, REM sleep-like state. This experimental model had not been previously used to study the neuropharmacological control of the upper airway. The results revealed a dose-dependent decrease in PCA muscle tone caused by pontine microinjections of carbachol. To investigate the cholinergic specificity of these effects, the muscarinic cholinergic antagonist pirenzepine was centrally administered before carbachol. Pirenzepine pretreatment effectively blocked the carbachol-induced, REM sleep-like state and attendant changes in muscle tone. These results specify for the first time that muscarinic cholinergic mechanisms within the pontine reticular formation can causally mediate state-dependent hypotonia in accessory respiratory muscles of the upper airway.
Microinjections of the cholinergic receptor agonist nicotine and the cholinesterase inhibitor neostigmine were made into the ventral tegmental area (VTA) of urethane-anesthetized rats, and dopamine (DA) efflux in the nucleus accumbens was measured using in vivo chronoamperometry. Dose-dependent increases in the chronoamperometric signals corresponding to increased DA efflux were observed in the nucleus accumbens of normal intact rats after cholinergic stimulation of the VTA. The source of the cholinergic input to the VTA was investigated by making excitotoxic lesions in either the laterodorsal tegmental nucleus (LDTg) or the pedunculopontine tegmental nucleus (PPTg). Compared with sham-operated control animals, which showed the same response as intact, nonlesioned rats, ibotenate lesions of the LDTg attenuated the stimulatory effects of intra-VTA neostigmine on DA efflux in the nucleus accumbens. In contrast, rats with ibotenate lesions of the PPTg showed normal nucleus accumbens DA eflux after intra-VTA injections of neostigmine. Such lesions in the PPTg attenuate DA efflux in the caudate-putamen stimulated by injections of neostigmine into the substantia nigra pars compacta (SNc). The present data show that cholinergic neurons in the LDTg, but not the PPTg, regulate the activity of DA-containing neurons in the VTA, which complements previous data showing that cholinergic neurons in the PPTg regulate DA-containing neurons in the SNc.
1. Whole cell recordings of glutamatergic excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation in the reticular formation were made from visualized hypoglossal motoneurons (HMs) in rat brain stem slices. 2. Carbachol, muscarine, or physostigmine reduced EPSC amplitude to 50 +/- 3%, 37 +/- 3%, and 54 +/- 7% (mean +/- SE) of control, respectively; effects of carbachol and physostigmine were antagonized by atropine (1-2 microM). EPSC depression was most effectively antagonized by methoctramine, an M2 muscarinic acetylcholine receptor (mAChR) antagonist with a high affinity constant (pKB) of 8.07 for the receptor mediating this response, whereas pirenzepine, an M1 mAChR antagonist, had a pKB of < 7.0, showing that EPSC depression was mediated by the M2 mAChR. 3. Postsynaptic properties of HMs (holding current and input resistance), EPSCs (reversal potential, rise time, half-width, and decay time constant), and postsynaptic glutamate-gated currents (amplitude and waveform) were not altered by carbachol or muscarine. 4. Muscarine did not decrease presynaptic neuron excitability, because the frequency of spontaneous EPSCs in HMs in the absence of tetrodotoxin (TTX) was either unchanged or increased. Leak and action currents of reticular formation neurons were not significantly altered by muscarine. In contrast, with TTX present, the frequency of spontaneous miniature glutamatergic EPSCs in HMs was decreased by both carbachol (mean change = 203 +/- 46%) and muscarine (mean change = 185 +/- 26%), with no change in miniature EPSC amplitude distribution. 5. Muscarinic depression of excitatory transmission to HMs thus occurs at the presynaptic terminal, most probably affecting release mechanisms downstream from calcium entry, and is likely to be significant during rapid eye movement sleep, possibly underlying the loss of tongue tone and inspiratory activity during this state.
Pontine cholinergic neurotransmission is known to play a key role in the regulation of rapid eye movement (REM) sleep and to contribute to state-dependent respiratory depression. Nitric oxide (NO) has been shown to alter the release of acetylcholine (ACh) in a number of brain regions, and previous studies indicate that NO may participate in the modulation of sleep/wake states. The present investigation tested the hypothesis that inhibition of NO synthase (NOS) within the medial pontine reticular formation (mPRF) of the unanesthetized cat would decrease ACh release, inhibit REM sleep, and prevent cholinergically mediated respiratory depression. Local NOS inhibition by microdialysis delivery of N(G)-nitro-L-arginine (NLA) significantly reduced ACh release in the cholinergic cell body region of the pedunculopontine tegmental nucleus and in the cholinoceptive mPRF. A second series of experiments demonstrated that mPRF microinjection of NLA significantly reduced the amount of REM sleep and the REM sleep-like state caused by mPRF injection of the acetylcholinesterase inhibitor neostigmine. Duration but not frequency of REM sleep epochs was significantly decreased by mPRF NLA administration. Injection of NLA into the mPRF before neostigmine injection also blocked the ability of neostigmine to decrease respiratory rate during the REM sleep-like state. Taken together, these findings suggest that mPRF NO contributes to the modulation of ACh release, REM sleep, and breathing.
Oromotor behavior results from the complex interaction between jaw, facial, and lingual muscles. The experiments in this and subsequent papers identify the sources of multisynaptic input to the trigeminal, facial, and hypoglossal motor nuclei. In the current experiments, pseudorabies virus (PRV-Ba) was injected into the jaw-opening (anterior digastric and mylohyoid) and jaw-closing muscles (masseter, medial pterygoid, and temporalis) in bilaterally sympathectomized rats. Injection volumes ranged from 2 to 21 mu l with average titers of 2.8 x 10(8) pfu/ml and maximum survival times of 96 h. The labeling patterns and distributions were consistent between each of the individual muscles and muscle groups. A predictable myotopic labeling pattern was produced in the trigeminal motor nucleus (Mo 5). Transneuronally labeled neurons occurred in regions known to project directly to Mo 5 motoneurons including the principal trigeminal sensory and supratrigeminal areas, Kolliker-Fuse region, nucleus subcoeruleus,and the parvicellular reticular formation. Maximum survival times revealed polysynaptic connections from the periaqueductal gray, laterodorsal and pedunculopontine tegmental areas, and the substantia nigra in the midbrain, ventromedial pontine reticular regions including the gigantocellular region and pars alpha and ventralis in the pens and medulla, and the nucleus of the solitary tract, paratrigeminal region, and paramedian field in the medulla. Thus, the results define the structure of the multisynaptic brainstem neural circuits controlling mandibular movement in the rat. (C) 1997 Elsevier Science B.V.
Cholinergic neurons in the mesopontine tegmentum are thought to play a critical role in the generation of paradoxical sleep (PS). However, no study has yet examined whether lesions of these neurons cause deficits of PS in the rat. We describe here the effects of lesions of the pedunculopontine tegmental nucleus (PPT) on spontaneous PS and on PS propensity, expressed during and after a short period of PS deprivation. Lesions were induced by bilateral injections of ibotenate. PS deprivation was performed manually by gently waking rats each time they showed polygraphic signs of PS. Two weeks after lesions, an 8-h baseline recording was performed; the following day, rats were PS deprived for 6 h and polygraphic recordings were then continued for 2 h, to examine recovery sleep. The same protocol was repeated 1 week later. Compared with controls and with rats with limited PPT lesions, rats bearing > 60% NADPH-diaphorase-positive cell loss within the PPT showed unaffected PS under baseline conditions. However, they made fewer attempts to enter PS during deprivation and they exhibited an attenuated rebound increase in PS time after deprivation. The number of PS attempts and the magnitude of PS rebound were negatively correlated with the percent loss of diaphorase-positive neurons within the PPT. Thus, PS propensity that accumulated as a result of PS deprivation was reduced after extensive PPT lesions. In summary, although spontaneous PS was found to be unaltered, the PS deprivation procedure used in this study demonstrated the dysfunctioning of PS caused by PPT lesions.
The NADPH-diaphorase histochemical technique provides a simple and robust method to stain select populations of neurons throughout the brain. We have recently identified the enzyme responsible for this histochemical reaction to be nitric oxide synthase. This enzyme is responsible for the calcium-dependent synthesis of nitric oxide from arginine. Nitric oxide acts as a novel neural messenger by stimulating soluble guanylyl cyclase thereby increasing the levels of cyclic guanosine 3',5'-monophosphate in target cells. Thus the NADPH-diaphorase histochemical method allows the direct visualization of the neurons which use this novel signal transduction pathway. We now describe the detailed distribution of this enzyme in the rat brain. Our results suggest a widespread role for the nitric oxide-cyclic guanosine monophosphate system in the nervous system.
Choline acetyltransferase immunhistochemistry was employed at light and electron microscopic levels in order to determine the distribution of cholinergic neurons in two subdivisions of the rat pedunculopontine tegmental nucleus that were previously defined on cytoarchitectonic grounds, and to compare the synaptic inputs to cholinergic and non-cholinergic somata in the subnucleus dissipatus, which receives major input from the substantia nigra. Large cholinergic neurons were found in both the pars compacta and the pars dissipata of the pedunculopontine nucleus. However, they were intermingled with non-cholinergic neurons and did not respect the cytoarchitectural boundaries of the nucleus. Ultrastructural study showed that all cholinergic neurons in the subnucleus dissipatus exhibited similar features. The majority had large somata (largest diameter ⩾20 μm) containing abundant cytoplasmic organelles and nuclei displaying a few shallow invaginations. Synaptic terminals on the cholinergic cell bodies were scarce and unlabeled boutons containing spherical synaptic vesicles and establishing asymmetric synaptic junctions were the dominant type. In contrast, the non-cholinergic neurons presented prominent differences in the size of their somata as well as in the distribution of axosomatic synapses. Two almost equally represented classes of non-cholinergic neurons which are referred to as large (largest diameter ⩾20 μm) and small (largest diameter
To clarify functional roles of mesopontine cholinergic neurons as a component of an activating system, single neuronal activity in the laterodorsal tegmental nucleus (LDT) of undrugged rats, whose head was fixed painlessly, was recorded along with cortical EEG and neck EMG. Activity of some dorsal raphe (DR) neurons was also recorded for comparison. Most of the animals had been sleep-deprived for 24 h. Observation was made only on neurons generating broad spikes, presumed from previous studies to be cholinergic or monoaminergic. The position of recorded neurons was marked by Pontamine sky blue ejected from the glass pipette microelectrode, and was identified on sections processed for NADPH diaphorase histochemistry which specifically stained cholinergic neurons. According to their firing rates during wakefulness (AW), slow-wave sleep (SWS) and paradoxical sleep (PS), 46 broad-spike neurons in the LDT were classified into 4 groups: (1) neurons most active during AW and silent during PS (some of these neurons might be serotonergic rather than cholinergic, as all the 9 neurons in the DR); (2) neurons most active during PS and silent during AW; (3) neurons equally more active during AW and PS than SWS; and (4) others mainly characterized by transiently facilitated activity at awakening and/or onset of PS. Neurons of groups 2 and 3 were the major constituents of the LDT. In most neurons change in firing preceded EEG change, except at awakening from PS. These results suggest that: (1) the LDT is composed of cholinergic neurons with heterogenous characteristics in relation to sleep/wakefulness; and (2) some tegmental cholinergic neurons play a privotal role in induction and maintenance of PS.
Descending brainstem projections from the pedunculopontine tegmental nucleus (PPN) were studied in the rat by use of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) and the retrograde tracer lectin-conjugated horseradish peroxidase (HRP-WGA). Results of these experiments demonstrated prominent bilateral projections to the pontomedullary reticular nuclei, but direct connections to the motor and sensory nuclei of the cranial nerves could not be ascertained. The PPN fibers terminated mainly in the pontine reticular nuclei oralis and caudalis and in ventromedial portions (pars alpha and pars ventralis) of the gigantocellular reticular nucleus. A smaller number of labeled fibers distributed to more dorsal regions of the gigantocellular nucleus, lateral para-gigantocellular, ventral reticular nucleus of the medulla and lateral reticular nucleus. Although a significant number of PHA-L labeled fibers was seen in two cases in the contralateral medial portion of the facial nucleus, and all cases exhibited a sparse predominantly ipsilateral projection to the lateral facial motor neurons, the retrograde tracing experiments have revealed that these facial afferents originated in the nuclei surrounding the PPN. The results are discussed in the context of PPN involvement in motor functions. It is suggested that the PPN may participate in a complex network involved in the orienting reflex.
The injection of carbachol into the pontine tegmentum of decerebrate cats evokes a postural motor atonia that has many of the characteristics of the atonia of natural rapid-eye-movement (REM) sleep (Morales et al. J. Neurophysiol. 57: 1118-1129, 1987). We have used the carbachol-injected decerebrate cat to study the changes in respiratory neuronal activity that accompany the atonia. The activities of representative respiratory motor nerves--phrenic, intercostal, and hypoglossal--and that of a motor branch of C4 were recorded in decerebrate, vagotomized, paralyzed, and artificially ventilated cats. After the microinjection of carbachol, there was a profound suppression of activity in all the nerves and a decrease in respiratory rate. This was a consistent stereotyped response in which the magnitude of the suppression of respiratory-related activity was phrenic (to approximately 65% of control) less than inspiratory intercostal (approximately 50%) less than hypoglossal (approximately 10%) less than expiratory intercostal (approximately 5%). The decrease in respiratory rate (to approximately 70% of control) was caused by a prolongation of both inspiratory and expiratory durations. Complete reversal of the carbachol effect was elicited by the microinjection of atropine into the same site as the carbachol injection. This allowed us to produce a second episode of atonia by the injection of carbachol into the contralateral pons. Thus we have demonstrated the existence of neural pathways originating in the cholinoceptive cells of the pons that have the potential to powerfully and differentially depress various respiratory motoneuronal pools and to reduce the respiratory rate. These pathways are likely to be activated along with the atonia of REM sleep.
Microinjections of the cholinergic agonist carbachol into a caudal part of the pontine reticular formation of the rat induce a rapid eye movement sleep-like state. This carbachol-sensitive region of the pontine reticular formation is innervated by cholinergic neurons in the pedunculopontine and laterodorsol tegmental nuclei. The same population of cholinergic neurons also project heavily to the thalamus, where there is good evidence that acetylcholine facilitates sensory transmission and blocks rhythmic thalamocortical activity. The present study was undertaken to examine the degree to which single cholinergic neurons in the mesopontine tegmentum project to both the carbachol-sensitive region of the pontine reticular formation and the thalamus, by combining double fluorescent retrograde tracing and immunofluorescence with a monoclonal antibody to choline acetyltransferase in the rat. The results indicated that a subpopulation (5-21% ipsilaterally) of cholinergic neurons in the mesopontine tegmentum projects to both the thalamus and the carbachol-sensitive site of the pontine reticular formation, and these neurons represented the majority (45-88%) of cholinergic neurons projecting to the pontine reticular formation site. The percentage of cholinergic neurons with dual projections was higher in the pedunculopontine tegmental nucleus (6-27%) than in the laterodorsal tegmental nucleus (4-11%). In addition, mixed with cholinergic neurons in the mesopontine tegmentum, there was a small population of dually projecting neurons that did not appear to be cholinergic. Mesopontine cholinergic neurons with dual projections may simultaneously modulate neuronal activity in the pontine reticular formation and the thalamus, and thereby have the potential of concurrently regulating different aspects of rapid eye movement sleep.
Acetylcholine (ACh) in the brain stem has been implicated in the generation of paradoxical sleep (PS). In order to clarify the relationship between local ACh release in the dorsal tegmental field (FTD), a possible PS-generating locus, and sleep-wake states in 6 cats. ACh was measured by the method of in vivo microdialysis and high performance liquid chromatography-electrochemical detection. It is noteworthy that ACh release was about 2 times higher (P less than 0.001) during PS than during slow-wave sleep and wakefulness in FTD, but not in the caudate nucleus, a control region. ACh release in FTD appeared to begin to increase prior to the onset of PS. Electrical and chemical (glutamate) stimulations of the nucleus magnocellularis (MC) enhanced ACh release in FTD and shortened PS latency. These results suggest that this PS-related enhancement of ACh release in FTD is induced by some cholinergic projections from glutamate-receptive neurons in MC.
The medullary and spinal efferents of the pedunculopontine tegmental nucleus and adjacent mesopontine tegmentum were investigated by employing (1) the anterograde autoradiographic methodology and (2) the retrograde transport of HRP and/or WGA-HRP in combination with choline acetyltransferase immunohistochemistry.
The anterograde experiments identified five descending pathways from the mesopontine tegmentum: (1) Probst's tract, which descends in the dorsolateral reticular formation in close relation to the nucleus of the solitary tract; (2) a ventrolateral branch of Probst's tract that extends ventrolaterally alongside the spinal trigeminal nucleus; (3) a ventromedial branch of Probst's tract that extends ventromedially through the gigantocellular field of the medulla; (4) the medial reticulospinal tract, which descends in parallel with the medial longitudinal fasciculus and turns ventrolaterally along the dorsal surface of the inferior olive to enter the ventrolateral funiculus of the spinal cord; and (5) a crossed ventromedial pathway, which descends in a ventral paramedian position through the magnocellular field of the medulla.
The origins of these pathways reflected a rough lateral-to-medial topography of mesopontine tegmental cell groups. The parabrachial nucleus, situated furthest laterally, for example, projected primarily through Probst's tract and its ventrolateral branch. The pedunculopontine tegmental nucleus, midbrain extrapyramidal area, and the subceruleal region, situated more medially, projected descending axons largely through the ventromedial branch of Probst's tract. The pontine tegmental field, situated furthest medially and ventromedially, was the largest contributor to the medial reticulospinal tract.
The retrograde transport experiments confirmed these general organizational features. The combination of retrograde transport with choline acetyltransferase immunohistochemistry established that the cholinergic pedunculopontine tegmental nucleus contributes a large portion to the mesopontine tegmental innervation of the medullary reticular formation. A much smaller number of cholinergic pedunculopontine neurons project as far as the spinal cord. Spinal projections from the mesopontine tegmentum originate largely from non-cholinergic neurons of the midbrain extrapyramidal area, subceruleal region, Kölliker-Fuse division of the parabrachial nucleus, and pontine tegmental field.
A total of 260 neurons were recorded in the rostral pontine tegmentum of freely moving cats during the sleep-waking cycle. Of these, 207 neurons (80%) were located in the dorsal pontine tegmentum containing monoaminergic and choline acetyltransferase (ChAT)-immunoreactive, or cholinergic neurons. In addition to presumably monoaminergic PS-off cells (n = 51) showing a cessation of discharge during paradoxical sleep (PS) and presumably cholinergic PGO-on cells (n = 40) exhibiting a burst of discharge just prior to and during ponto-geniculo-occipital (PGO) waves, we observed tonic (n = 108) and phasic (n = 61) neurons exhibiting, respectively, tonic and phasic patterns of discharge during wakefulness and/or paradoxical sleep. Of 87 tonic cells histologically localized in the dorsal pontine tegmentum rich in cholinergic neurons, 46 cells (53%) were identified as giving rise to ascending projections either to the intralaminar thalamic complex (n = 26) or to the ventrolateral posterior hypothalamus (n = 13) or to both (n = 9). Two types of tonic neurons were distinguished: 1) tonic type I neurons (n = 28), showing a tonic pattern and high rates of discharge during both waking and paradoxical sleep as compared with slow wave sleep; and 2) tonic type II neurons (n = 20), exhibiting a tonic pattern of discharge highly specific to the periods of paradoxical sleep. Tonic type I neurons were further divided into two subclasses on the basis of discharge rates during waking: a) rapid (Type I-R; n = 17); and b) slow (Type I-S; n = 11) units with a discharge frequency of more than 12 spikes/s or less than 5 spikes/s, respectively. Like monoaminergic PS-off and cholinergic PGO-on cells, both tonic type II and type I-S cells were characterized by a long spike duration (median: 3.3 and 3.5 ms), as well as by a slow conduction velocity (median = 1.8 and 1.7 m/s). In the light of these data, we discuss the possible cholinergic nature and functional significance of these ascending tonic neurons in the generation of neocortical electroencephalographic desynchronization occurring during waking and paradoxical sleep.
Descending projections from cholinergic neurons in the pedunculopontine and laterodorsal tegmental nuclei, collectively referred to as the pontomesencephalotegmental (PMT) cholinergic complex, were studied by use of the fluorescent retrograde tracers fluorogold, true blue, or Evans Blue in combination with choline acetyltransferase (ChAT) immunohistochemistry of acetylcholinesterase (AChE) pharmacohistochemistry. Pedunculopontine somata positive for ChAT or staining intensely for AChE were retrogradely labeled with fluorescent tracers following infusions into the motor nuclei of cranial nerves 5, 7, and 12. ChAT-positive cells in both the pedunculopontine and laterodorsal tegmental nuclei demonstrated projections to the vestibular nuclei, the spinal nucleus of the 5th cranial nerve, deep cerebellar nuclei, pontine nuclei, locus ceruleus, raphe magnus nucleus, dorsal raphe nucleus, median raphe nucleus, the medullary reticular nuclei, and the oral and caudal pontine reticular nuclei. Fluorescent tracers used in combination with AChE pharmacohistochemistry corroborated these projections and, in addition, provided evidence for cholinergic pontomesencephalic projections to the lateral reticular nucleus and inferior olive. The majority of retrogradely labeled neurons demonstrating ChAT-like immunoreactivity were found ipsilateral to the injection site, but, in all cases, tracer-containing cholinergic cells contralateral to the infused side of the brain were detected also. More retrogradely labeled cells containing ChAT were observed in the pedunculopontine tegmental than in the laterodorsal tegmental nucleus following tracer injections at all sites with the exceptions of the locus ceruleus and dorsal raphe nucleus where the converse profile was observed. None of the pedunculopontine or laterodorsal tegmental cells immunopositive for ChAT or stained intensely for AChE contained retrogradely transported tracers following dye infusions into the cerebellar cortex or cervical spinal cord. Triple-label experiments using two tracers infused into different sites in the same animal revealed that individual ChAT-immunoreactive cells in the PMT cholinergic complex projected to more than one hindbrain site in some cases and had ascending projections as well. Certain ChAT-positive somata in the pedunculopontine and laterodorsal tegmental nuclei were found in close association with several fiber tracts, including the superior cerebellar peduncle, lateral lemniscus, dorsal tegmental tract, and medial longitudinal fasciculus.
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.
This study demonstrates that the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) are sources of cholinergic projections to the cat pontine reticular formation gigantocellular tegmental field (PFTG). Neurons of the LDT and PPT were double-labeled utilizing choline acetyltransferase immunohistochemistry combined with retrograde transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). In the LDT the percentage of cholinergic neurons retrogradely labeled from PFTG was 10.2% ipsilaterally and 3.7% contralaterally, while in the PPT the percentages were 5.2% ipsilaterally and 1.3% contralaterally. These projections from the LDT and PPT to the PFTG were confirmed and their course delineated with anterograde labeling utilizing Phaseolus vulgaris leucoagglutinin (PHA-L) anterograde transport.
The ascending cholinergic projections of the pedunculopontine and dorsolateral tegmental nuclei, referred to collectively as the pontomesencephalotegmental (PMT) cholinergic complex, were investigated by use of fluorescent tracer histology in combination with choline-O-acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. Propidium iodide, true blue, or Evans blue was infused into the anterior, reticular, mediodorsal, central medial, and posterior nuclear areas of the thalamus; the habenula; lateral geniculate; superior colliculus; pretectal/parafascicular area; subthalamic nucleus; caudate-putamen complex; globus pallidus; entopeduncular nucleus; substantia nigra; medial septal nucleus/vertical limb of the diagonal band area; magnocellular preoptic/ventral pallidal area; and lateral hypothalamus. In some animals, separate injections of propidium iodide and true blue were made into two different regions in the same rat brain, usually a dorsal and a ventral target, in order to assess collateralization patterns. Retrogradely transported fluorescent labels and ChAT and/or AChE were analyzed microscopically on the same brain section. All of the above-delimited targets were found to receive cholinergic input from the PMT cholinergic complex, but some regions were preferentially innervated by either the pedunculopontine or dorsolateral tegmental nucleus. The former subdivision of the PMT cholinergic complex projected selectively to extrapyramidal structures and the superior colliculus, whereas the dorsolateral tegmental nucleus was observed to provide cholinergic input preferentially to anterior thalamic regions and rostral portions of the basal forebrain. The PMT cholinergic neurons showed a tendency to collateralize extensively.
Projections to the trigeminal, facial, ambiguus, and hypoglossal motor nuclei were determined by using horseradish peroxidase histochemistry. Most of the afferent projections to these motor nuclei were from the brainstem reticular formation, frequently in areas adjacent to other synergetic motor nuclei. The reticular formation lateral to the hypoglossal nucleus and reticular structures surrounding the trigeminal motor nucleus projected to each of these other brainstem motor nuclei involved in oral-facial function. Afferent projections to these motor nuclei also were organized along the rostrocaudal axis. Within the reticular formation most of the afferent projections to the trigeminal motor nucleus originated rostral to the majority of neurons projecting to the hypoglossal and ambiguus nuclei, which in turn were rostral to the primary source of reticular afferents to the facial nucleus. In comparison, projections from the sensory trigeminal nuclei and nucleus of the solitary tract were sparse. The interneuron pools that project to the orofacial motoneurons provide one further link in understanding the brainstem substrates for integrating oral and ingestive behaviors.
Monoclonal antibodies to choline acetyltransferase and a histochemical method for the concurrent demonstration of acetylcholinesterase and horseradish peroxidase were used to investigate the organization of ascending cholinergic pathways in the central nervous system of the rat. The cortical mantle, the amygdaloid complex, the hippocampal formation, the olfactory bulb and the thalamic nuclei receive their cholinergic innervation principally, from cholinergic projection neurons of the basal forebrain and upper brainstem. On the basis of connectivity patterns, we subdivided these cholinergic neurons into six major sectors. The Ch1 and Ch2 sectors are contained within the medial septal nucleus and the vertical limb nucleus of the diagonal band, respectively. They provide the major cholinergic projections of the hippocampus. The Ch3 sector is contained mostly within the lateral portion of the horizontal limb nucleus of the diagonal band and provides the major cholinergic innervation to the olfactory bulb. The Ch4 sector includes cholinergic neurons in the nucleus basalis, and also within parts of the diagonal band nuclei. Neurons of the Ch4 sector provide the major cholinergic innervation of the cortical mantle and the amygdala. The Ch5-Ch6 sectors are contained mostly within the pedunculopontine nucleus of the pontomesencephalic reticular formation (Ch5) and within the laterodorsal tegmental gray of the periventricular area (Ch6). These sectors provide the major cholinergic innervation of the thalamus. The Ch5-Ch6 neurons also provide a minor component of the corticopetal cholinergic innervation. These central cholinergic pathways have been implicated in a variety of behaviors and especially in memory function. It appears that the age-related changes of memory function as well as some of the behavioral disturbances seen in the dementia of Alzheimer's Disease may be related to pathological alterations along central cholinergic pathways.
The neuroanatomical location and cytological features of cholinergic neurons in the rat brain were determined by the immunocytochemical localization of the biosynthetic enzyme, choline acetyltransferase (ChAT). Perikarya labeled with ChAT were detected in four major cell groups: (1) the striatum, (2) the magnocellular basal nucleus, (3) the pontine tegmentum, and (4) the cranial nerve motor nuclei. Labeled neurons in the striatum were observed scattered throughout the neostriatum (caudate, putamen) and associated areas (nucleus accumbens, olfactory tubercle). Larger ChAT-labeled neurons were seen in an extensive cell system which comprises the magnocellular basal nucleus. This more or less continuous set of neuronal clusters consists of labeled neurons in the nucleus of the diagonal band (horizontal and vertical limbs), the magnocellular preoptic nucleus, the substantia innominata, and the globus pallidus. Labeled neurons in the pontine tegmentum were seen as a group of large neurons in the caudal midbrain, dorsolateral to the most caudal part of the substantia nigra, and extended in a caudodorsal direction through the midbrain reticular formation into the area surrounding the superior cerebellar peduncle. The neurons in this latter group constitute the pedunculopontine tegmental nucleus (PPT). An additional cluster of cells was observed medially adjacent to the PPT, in the lateral part of the central gray matter at the rostral end of the fourth ventricle. This group corresponds to the laterodorsal tegmental nucleus. Large ChAT-labeled neurons were also observed in all somatic and visceral motor nerve nuclei. The correspondence of the distribution of ChAT-labeled neurons identified by our methods to earlier immunocytochemical and acetylcholinesterase histochemical studies and to connectional studies of these groups argues for the specificity of the ChAT antibody used.
Nitric oxide synthase immunoreactivity was detected in neurons and fibers of the rat pontine medulla. In the medulla, nitric oxide synthase-positive neurons and processes were observed in the gracile nucleus, spinal trigeminal nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, nucleus ambiguus, medial longitudinal fasciculus, reticular nuclei and lateral to the pyramidal tract. In the pons, intensely labeled neurons were observed in the pedunuclopontine tegmental nucleus, paralemniscal nucleus, ventral tegmental nucleus, laterodorsal tegmental nucleus, and lateral and medial parabrachial nuclei. Labeled neurons and fibers were seen in the interpeduncular nuclei, dorsal and median raphe nuclei, central gray and dorsal central gray, and superior and inferior colliculi. Double-labeling techniques showed that a small population (<5%) of nitric oxide synthase-positive neurons in the medulla also contained immunoreactivity to the aminergic neuron marker tyrosine hydroxylase. The majority of nitric oxide synthase-immunoreactive neurons in the dorsal and median raphe nuclei were 5-hydroxytryptamine-positive, whereas very few 5-hydroxytryptamine-positive cells in the caudal raphe nuclei were nitric oxide synthase-positive. Virtually all nitric oxide synthase-positive neurons in the pedunuclopontine and laterodorsal tegmental nuclei were also choline acetyltransferase-positive, whereas nitric oxide synthase immunoreactivity was either low or not detected in choline acetyltransferase-positive neurons in the medulla.
Cholinergic mechanisms are known to play a key role in the regulation of breathing, but the distribution of muscarinic receptor (mAChR) subtypes has not been localized within brain stem respiratory nuclei. This study examined the hypothesis that mAChR subtypes are heterogeneously distributed across brain stem nuclei that control breathing. With the use of in vitro receptor autoradiography, the results provide the first selective labeling and quantitative mapping of M1, M2, and M3 mAChR subtypes in cat brain stem regions known to regulate breathing. Among brain stem nuclei known to contain respiratory-related neurons, the greatest amount of mAChR binding was measured in the lateral and medial parabrachial nuclei and the lateral nucleus of the solitary tract. Fewer mAChRs were localized in nuclei comprising the ventral respiratory group (nucleus ambiguous, retrofacial nucleus) and ventral medulla (retrotrapezoid nucleus and ventrolateral medulla). The data provide an essential first step for future studies aiming to specify the regulatory role of mAChR subtypes within brain stem respiratory nuclei.
Protrusion and retraction of the tongue are essential components of such orofacial behaviors as mastication, respiration, and swallowing. Stimulation of the medial branch of the hypoglossal nerve yields tongue protrusion, while stimulation of the lateral branch yields tongue retraction in rat. We exploited the transsynaptic transport capabilities of pseudorabies virus to determine specific circuits that innervate protruder and retractor muscles of the rat tongue. Each group of muscles is innervated by distinct populations of hypoglossal motoneurons: caudal ventral and ventrolateral motoneurons form the largest proportion of those innervating protruders, whereas rostral dorsal motoneurons innervate retractors. Our primary finding was differential innervation of protruder and retractor motoneurons by premotoneurons in the lateral tegmental field: premotoneurons innervating protruder motoneurons were more ventral and ventromedial than those innervating retractor motoneurons. In addition, protruder motoneurons received projections from the ipsilateral lateral parabrachial nucleus but not spinal trigeminal nucleus or medial and ventral subnuclei of the solitary tract; the converse was true for retractor motoneurons. These results suggest segregation of functional networks that control hypoglossal motoneurons. The dorsal medulla, in or around the solitary tract, contains neurons specific to retractor motoneurons, and the region ventrolateral to the hypoglossal nucleus contains circuitry specific to protruder motoneurons. Common innervation of medial and lateral branch motoneurons is provided by premotoneurons in the raphe and gigantocellular reticular formation of the medial medulla. The midline medullary nuclei with diverse projections may coordinate complex behavior or modulate general motoneuron excitability, whereas the lateral reticular formation, with anatomically discrete projections, may control motoneurons that contribute to distinct orofacial behaviors.
A group of medium-to-large cholinergic neurons situated in the dorsolateral mesopontine tegmentum comprises the pedunculopontine tegmental nucleus (PPT). The PPT pars compacta (PPT-pc), which occupies the lateral part of the caudal two-thirds of the nucleus, contains a dense aggregation of cholinergic neurons. In the present study, we have employed immunohistochemistry for choline acetyltransferase (ChAT) and electron microscopy to investigate the ultrastructure and synaptic organization of neuronal elements in the PPT-pc. Our results demonstrate that: (1) ChAT-immunoreactive (i.e., cholinergic) PPT-pc neurons are characterized by abundant cytoplasm and organelles, and have few axosomatic synapses (both asymmetric and symmetric); (2) ChAT-immunoreactive dendrites comprise 6-15% of total dendritic elements in the neuropil; the mean percentage of dendritic membrane covered by synaptic terminals is approximately 15%, and nearly all synapses with ChAT-immunoreactive dendrites are asymmetric; (3) within the boundaries described by cholinergic PPT-pc, there are noncholinergic neurons which, in contrast, exhibit a lucent cytoplasm and a higher frequency of axosomatic synapses (10.5% versus 3.7% for cholinergic neurons); and (4) noncholinergic neurons are morphologically heterogeneous with one subpopulation exhibiting a mean diameter that approximates that of cholinergic cells (i.e., > 15 microns and < 20 microns) and a very high frequency of axosomatic synapses (> 20%). Only 0.2-0.7% of terminal elements in the neuropil were ChAT-immunoreactive and these were not observed to synapse with cholinergic dendrites or somata. This relative paucity of terminal labeling and lack of cholinergic-cholinergic interactions seems inconsistent with the recognized and prominent physiological actions of acetylcholine on cholinergic PPT-pc neurons, and suggests a methodological limitation and/or a potential paracrine-like action of nonsynaptically released acetylcholine in the PPT region.
The present experiments continue our investigations of the higher order afferent systems controlling the orofacial musculature. Pseudorabies virus (PRV) was injected into the buccinator, platysma, posterior digastric, and zygomatic muscles in bilaterally sympathectomized rats. Injection volumes ranged from 6 to 12 microl with average titers of 7 x 10(8) pfu/ml and maximum survival times of 96 h. The labeling patterns and distributions were similar across the individual muscles and between muscle groups (perioral vs. posterior digastric), as well as in comparison to the results from previous masticatory muscle injections. Injections produced a predictable myotopic labeling pattern in the facial motor nucleus (Mo 7) and transneuronally in regions known to project directly to Mo 7 including the red nucleus, ventrolateral parabrachial region, principal trigeminal sensory nucleus, supratrigeminal area, and the parvicellular reticular formation. Maximum survival times revealed more distant connections from a variety of nuclear zones including the periaqueductal gray, laterodorsal and pedunculopontine tegmental areas, and the substantia nigra in the midbrain, ventromedial reticular regions including the gigantocellular region and pars alpha and ventralis in the pons and medulla, and the nucleus of the solitary tract, spinal trigeminal nucleus caudalis, paratrigeminal region, and paramedian field in the medulla. The similarity of the labeling patterns and distributions of the higher order afferents resulting from PRV facial and masticatory muscle injections identifies the neural circuits that may coordinate the activity of these muscle groups during oral motor behavior.
Retrograde and anterograde axonal transport techniques were used to investigate the organization of inputs from the dorsomedial medulla, a region known to elicit patterned swallowing reflexes following focal stimulation, to the fifth (MoV), seventh (VII), tenth (nucleus ambiguus, NA), and twelfth (XII) cranial nerve motor nuclei in the rat, those motor nuclei most directly involved in the control of deglutition. The results may be summarized as follows. 1) Dorsal medullary inputs to MoV, VII, and XII arise primarily from an extended region of the caudal reticular formation immediately ventral to the nucleus of the solitary tract (NTS), which we term the dorsal medullary reticular column (DMRC). Projections from the DMRC are largely bilateral and are distributed preferentially to the ventral subdivision of MoV, to the dorsal and intermediate subdivisions of VII, and to both the dorsal and the ventral subdivisions of XII. In addition, a subpopulation of large multipolar neurons embedded within the DMRC gives rise to a primarily crossed input to the dorsal subdivision of MoV. 2) Dorsal medullary inputs to the NA arise from the NTS, are largely uncrossed, and are organized such that the ventrolateral, intermediate, and interstitial subdivisions of the NTS project to the semicompact formation and to the rostral extension of the compact formation (which supplies the pharynx) and to the loose formation (larynx), whereas the central subdivision of the NTS provides input to the compact formation (esophagus). 3) Neither the NTS nor the DMRC gives rise to significant projections to the central subnucleus of the NTS. Together, these results provide evidence for discrete medullary pathways subserving sequential activation of swallowing reflexes.
In individuals with compromised upper airway anatomy, genioglossus (GG), the main protruder muscle of the tongue, is an important upper airway dilator which helps prevent upper airway obstructions. During rapid eye movement (REM) sleep, both the tonic and inspiratory-modulated components of GG activity are suppressed in parallel with the characteristic postural atonia. We tested whether the REM sleep-related reduction in the respiratory activity of GG may, in part, result from a reduced inspiratory drive relayed to hypoglossal (XII) motoneurons from their premotor medullary inspiratory neurons. In 15 urethane-anesthetized, paralyzed, vagotomized and artificially ventilated rats, we recorded XII nerve activity and the extracellular activity of medullary inspiratory-modulated neurons antidromically activated with latencies of 0.8 ms +/- 0.3(SD) from within (n = 19) or adjacent to (n = 11) the XII nucleus. Carbachol (10-20 nl, 10 mM), a cholinergic agonist, was microinjected into the dorsomedial pons. Such injections trigger a REM sleep-like state in chronically instrumented, intact animals and, in anesthetized rats, produce respiratory and electrocortical changes similar to those of REM sleep. Following the injections, the respiratory component of XII nerve activity was depressed by 51 +/- 22%, while the mean inspiratory firing rate of the neurons decreased by only 7.4 +/- 13.8% (from 69 +/- 34 Hz to 65 +/- 37 Hz; P < 0.02; n = 30). The activity of ventral respiratory group (VRG) and reticular formation inspiratory neurons with axons within the XII nucleus was reduced by 10 +/- 14% (P < 0.005; n = 19), whereas the activity of neurons located near the VRG that had axons passing below the XII nucleus did not change (n = 5). Thus, the depressant effect of carbachol on medullary inspiratory neurons was slightly more pronounced in reticular formation and VRG cells premotor to XII motoneurons than in other medullary inspiratory cells. For all cells, the magnitude of the decrease of cell activity was not related to the magnitude of depression of XII nerve activity, the simultaneously occurring decrease in respiratory rate or the cell's control firing rate. Since the magnitude of this depressant effect on all cell types was disproportionately small when compared with the depression of XII nerve activity, the REM sleep-like decrease in GG activity must be mainly mediated by non-respiratory premotor pathways.
Hypoglossal motoneurons (HMNs), which innervate the tongue muscles, are involved in several important physiological functions, including the maintenance of upper airway patency. The neural mechanisms that affect HMN excitability are therefore important determinants of effective breathing. Obstructive sleep apnea is a disorder characterized by recurrent collapse of the upper airway that is likely due to decline of pharyngeal motoneuron activity during sleep. Because cholinergic neuronal activity is closely coupled to wake and sleep states, we tested the effects and pharmacology of nicotinic acetylcholine receptor (nAChR) activation on HMNs. We made intracellular recordings from HMNs in medullary slices from neonatal rats and found that local application of the nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, excited HMNs by a Ca(2+)-sensitive, and TTX-insensitive inward current that was blocked by dihydro-beta-erythroidine (IC(50): 19+/-3 nM), methyllycaconitine (IC(50): 32+/-7 nM), and mecamylamine (IC(50): 88+/-11 nM), but not by alpha-bungarotoxin (10 nM). This is consistent with responses being mediated by postsynaptic nAChRs that do not contain the alpha7 subunit. These results suggest that nAChR activation may contribute to central maintenance of upper airway patency and that the decline in firing rate of cholinergic neurons during sleep could potentially disfacilitate airway dilator muscle activity, contributing to airway obstruction.
The effects on serotoninergic, noradrenergic and cholinergic markers on neurons of the pontomesencephalic tegmentum nuclei were studied in rats following local administration of fibrillar beta-amyloid peptide (Abeta1-40) into the left retrosplenial cortex. Focal deposition of Abeta in the retrosplenial cortex resulted in a loss of serotoninergic neurons in the dorsal and median raphe nuclei. The dorsal raphe nucleus showed a statistically significant reduction of 31.7% in the number of serotoninergic neurons and a decrease (up to 17.38%) in neuronal density in comparison with the same parameters in uninjected controls. A statistically significant reduction of 50.3%, together with a significant decrease of 53.94% in the density of serotoninergic neurons, was also observed in the median raphe nucleus as compared with control animals. Furthermore, a significant reduction of 35.07% in the number of noradrenergic neurons as well as a statistically significant decrease of 56.55% in the density of dopamine-beta-hydroxylase-immunoreactive neurons were also found in the locus coeruleus as compared with the corresponding hemisphere in uninjected controls. By contrast, a reduction of 24.37% in the number of choline acetyltransferase-positive neurons and a slight decrease (up to 22.28%) in the density of cholinergic neurons, which were not statistically significant, was observed in the laterodorsal tegmental nucleus in comparison with the same parameters in control animals. These results show that three different neurochemically defined populations of neurons in the pontomesencephalic tegmentum are affected by the neurotoxicity of Abeta in vivo and that Abeta might indirectly affect serotoninergic, noradrenergic and cholinergic innervation in the retrosplenial cortex.
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.
The genioglossus (GG) muscle of the tongue, innervated by the hypoglossal motor nucleus (HMN), helps maintain an open airway for effective breathing. In vitro studies in neonatal rodents have separately characterized muscarinic and nicotinic receptor influences at the HMN but the net effects of combined nicotinic and muscarinic receptor activation and increased endogenous acetylcholine have not been determined in adult animals in vivo. Urethane-anaesthetized, tracheotomized and vagotomised rats were studied. Microdialysis perfusion of acetylcholine into the HMN significantly decreased respiratory-related GG activity (28.5 +/- 11.0% at a threshold dose of 0.1 mm). Application of the cholinergic agonists carbachol and muscarine have similar suppression effects (GG activity was decreased 11.8 +/- 4.3 and 20.5 +/- 5.8%, respectively, at 0.01 microm). Eserine, an acetylcholinesterase inhibitor, also decreased the amplitude of respiratory-related GG activity (36.4 +/- 11.3% at 1.0 microm) indicating that endogenous acetylcholine modulates GG activity. Although these results showed that suppression of GG activity predominates during cholinergic stimulation at the HMN, application of the nicotinic receptor agonist dimethyl-4-phenylpiperazinium iodide significantly increased tonic and respiratory-related GG activity (156 +/- 33% for respiratory activity at 1.0 mm) showing that excitatory responses are also present. Consistent with this, 100 microm carbachol decreased GG activity by 44.2 +/- 7.5% of control, with atropine (10 microm) reducing this suppression to 13.8 +/- 4.0% (P < 0.001). However, the nicotinic receptor antagonist dihydro-beta-erythroidine (100 microm) increased the carbachol-mediated suppression to 69.5 +/- 5.9% (P = 0.011), consistent with a role for nicotinic receptors in limiting the overall suppression of GG activity during cholinergic stimulation. Application of eserine to increase endogenous acetylcholine also showed that inhibitory muscarinic and excitatory nicotinic receptors together determine the net level of GG activity during cholinergic stimulation at the HMN. The results suggest that acetylcholine has mixed effects at the HMN with muscarinic-mediated GG suppression masking nicotinic excitation.
Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch1-Ch6)
- Mm Mesulam
- Ej Mufson
- Bh Wainer
- Ai Levey
Mesulam MM, Mufson EJ, Wainer BH, Levey AI. Central cholinergic pathways in the rat: an
overview based on an alternative nomenclature (Ch1-Ch6). Neuroscience 1983;10:1185–1201.
[PubMed: 6320048]
Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch1–Ch6)
- Mesulam
Histochemical mapping of nitric oxide synthase in the rat brain
- Vincent