No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Abstract
Synaptic connectivity and other ultrastructural features of cholinergic and non-cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei were investigated with electron microscopy combined with pre-embedding immunohistochemistry for choline acetyltransferase. Quantitative morphometric analyses were conducted on selected immunopositive as well as immunonegative neurons. The ultrastructure of immunoreactive neurons in the laterodorsal and pedunculopontine tegmental nuclei was similar. In both nuclei, immunoreactive neurons were among the larger neurons, and somatic areas of immunopositive neurons in single thin sections were larger than those of immunonegative neurons by an average of 40%. Immunopositive somata varied in shape, appearing polygonal, fusiform or oval. Regardless of immunoreactivity, however, neurons in the pedunculopontine nucleus tended to have more irregular shapes than those in the laterodorsal tegmental nucleus. Immunoreactive neurons in both the nuclei had abundant cytoplasmic organelles and a large, clear nucleus with a few infoldings. Usually, about a quarter of the surface of an immunopositive soma was covered with astrocytic processes, and some immunopositive somata were directly apposed to an astrocyte. Immunoreactive dendrites and, less frequently, axon terminals were seen in close apposition to endothelial cells of blood capillaries or pericytes. Immunoreactive somata and dendrites in the laterodorsal and pedunculopontine tegmental nuclei received many synapses, mainly from unlabelled axon terminals. The mean number (4.7 +/- 1.8) of synapses received by immunolabelled somata in single thin sections was greater, by about 70%, than those received by unlabelled somata. The presynaptic axon terminals synapsing with immunoreactive somata commonly contained small, round and clear vesicles, and 20% of them contained a few dense-cored vesicles as well. Immunoreactive dendrites, in addition, received synapses from unlabelled axon terminals containing flat and clear vesicles, which accounted for 15% of the synapses with immunoreactive dendrites. Many immunopositive axon terminals were present in both the tegmental nuclei. They contained clear round vesicles, and usually synapsed with unlabelled dendrites. A few immunolabelled axons, however, appeared to synapse with immunopositive somata and dendrites. Immunoreactive fibres were also present in both the tegmental nuclei. They were either thinly myelinated or unmyelinated. In conclusion, the ultrastructural morphology of cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei is similar, and these neurons represent a distinct population of neurons in both nuclei in that they are larger and receive more synaptic contacts than non-cholinergic neurons. Cholinergic neurons, however, appear to receive synapses from cholinergic axon terminals only rarely, despite the abundance of cholinergic terminals in the tegmental nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)
... The LDT contains predominantly cholinergic, glutamatergic, and GABAergic neurons and is known as a part of brainstem cholinergic system 15 . It has been indicated that the LDT contains CRF-and urocortin (Ucn1)-containing neurons [16][17][18] , which are involved in the stress-associated progression of anxiety, depression, SUD, inappropriate arousal, and control of the sleep and wakefulness [19][20][21][22][23] . These neurons contribute to the elevated risk of psychiatric disorders linked to chronic stress by modulating the cholinergic transmission of LDT 24 . ...
... The LDT-VTA pathway plays a critical role in regulating the activity of VTA DA neurons and the development of motivated behaviors 28,31 . It has been indicated that LDT regulates stress through CRF-and Ucn1-containing neurons [16][17][18] , which are involved in the development of stress-associated disorders, including SUD, inappropriate arousal, anxiety, and depression [19][20][21] . The high activity of cholinergic neurotransmission has also been shown to be involved in the progression of psychiatric disorders 34,35 . ...
Exposure to prenatal stress (PS) leads to the offspring's vulnerability towards the development of cognitive and behavioral disorders. Laterodorsal tegmentum (LDT) is a part of the brainstem cholinergic system that is believed to play a pivotal role in the stress-associated progression of anxiety, memory impairment, and addictive behaviors. In this study, we aimed to investigate the electrophysiological alterations of LDT cholinergic neurons and its accompanied behavioral and cognitive outcomes in the offspring of mice exposed to physical or psychological PS. Swiss Webster mice were exposed to physical or psychological stress on the tenth day of gestation. Ex vivo investigations in LDT brain slices of adolescent male offspring were performed to evaluate the effects of two stressor types on the activity of cholinergic neurons. Open field test, elevated plus maze, passive avoidance test, and conditioned place preference were conducted to assess behavioral and cognitive alterations in the offspring. The offspring of both physical and psychological PS-exposed mice exhibited increased locomotor activity, anxiety-like behavior, memory impairment, and preference to morphine. In both early- and late-firing cholinergic neurons of the LDT, stressed groups demonstrated higher firing frequency, lower adaptation ratio, decreased action potential threshold, and therefore increased excitability compared to the control group. The findings of the present study suggest that the hyperexcitability of the cholinergic neurons of LDT might be involved in the development of PS-associated anxiety-like behaviors, drug seeking, and memory impairment.
... One of the main mesopontine cholinergic areas is the pedunculopontine nucleus (PPN), which is not only the source of cholinergic fibers but also receives cholinergic inputs from the neighboring laterodorsal tegmental nucleus (LDT), the contralateral PPN, and local cholinergic axon collaterals (Honda and Semba, 1995;Mena-Segovia et al., 2008). The PPN has cholinergic and non-cholinergic (GABAergic and glutamatergic) neurons, which show different activity patterns during global brain states, such as slow-wave sleep (SWS), paradoxical sleep (PS), and wakefulness (W). ...
... Taken together, it seems likely that cholinergic activation of a cholinergic nucleus desynchronizes its neuronal population. As the PPN provides local axon collaterals for itself, innervates the contralateral PPN, and receives cholinergic fibers from the LDT, cholinergic activation might spread to all mesopontine cholinergic structures and contribute to the desynchronization of cholinergic neuronal populations (Honda and Semba, 1995;Mena-Segovia et al., 2008). Desynchronization of PPN units takes place in parallel with cortical desynchronization (Mena-Segovia et al., 2008;Boucetta et al., 2014;Petzold et al., 2015). ...
The pedunculopontine nucleus (PPN), a structure known as a cholinergic member of the reticular activating system (RAS), is source and target of cholinergic neuromodulation and contributes to the regulation of the sleep–wakefulness cycle. The M-current is a voltage-gated potassium current modulated mainly by cholinergic signaling. KCNQ subunits ensemble into ion channels responsible for the M-current. In the central nervous system, KCNQ4 expression is restricted to certain brainstem structures such as the RAS nuclei. Here, we investigated the presence and functional significance of KCNQ4 in the PPN by behavioral studies and the gene and protein expressions and slice electrophysiology using a mouse model lacking KCNQ4 expression. We found that this mouse has alterations in the adaptation to changes in light–darkness cycles, representing the potential role of KCNQ4 in the regulation of the sleep–wakefulness cycle. As cholinergic neurons from the PPN participate in the regulation of this cycle, we investigated whether the cholinergic PPN might also possess functional KCNQ4 subunits. Although the M-current is an electrophysiological hallmark of cholinergic neurons, only a subpopulation of them had KCNQ4-dependent M-current. Interestingly, the absence of the KCNQ4 subunit altered the expression patterns of the other KCNQ subunits in the PPN. We also determined that, in wild-type animals, the cholinergic inputs of the PPN modulated the M-current, and these in turn can modulate the level of synchronization between neighboring PPN neurons. Taken together, the KCNQ4 subunit is present in a subpopulation of PPN cholinergic neurons, and it may contribute to the regulation of the sleep–wakefulness cycle.
... It was demonstrated that these neurons have greater complexity and abundance of cellular processes, with dendrites that can reach up to 300 µm in length, which allows them to create a morphological substrate on which multiple connections can be established (Aitken et al. 2017). An interesting aspect is the great interconnection between the cholinergic and the non-cholinergic populations in the PPTg (Honda and Semba 1995). Some authors even suggest that there is a similarity between the SN and PPTg regarding the close relationship of their three types of populations (Honda and Semba 1995), to the point that some refer to the PPTg as an extension of the SN (Di Chiara et al. 1979;Gulcebi et al. 2012;Hormigo et al. 2016). ...
... An interesting aspect is the great interconnection between the cholinergic and the non-cholinergic populations in the PPTg (Honda and Semba 1995). Some authors even suggest that there is a similarity between the SN and PPTg regarding the close relationship of their three types of populations (Honda and Semba 1995), to the point that some refer to the PPTg as an extension of the SN (Di Chiara et al. 1979;Gulcebi et al. 2012;Hormigo et al. 2016). ...
The acoustic startle reflex (ASR) is a short and intense defensive reaction in response to a loud and unexpected acoustic stimulus. In the rat, a primary startle pathway encompasses three serially connected central structures: the cochlear root neurons, the giant neurons of the nucleus reticularis pontis caudalis (PnC), and the spinal motoneurons. As a sensorimotor interface, the PnC has a central role in the ASR circuitry, especially the integration of different sensory stimuli and brain states into initiation of motor responses. Since the basal ganglia circuits control movement and action selection, we hypothesize that their output via the substantia nigra (SN) may interplay with the ASR primary circuit by providing inputs to PnC. Moreover, the pedunculopontine tegmental nucleus (PPTg) has been proposed as a functional and neural extension of the SN, so it is another goal of this study to describe possible anatomical connections from the PPTg to PnC. Here, we made 6-OHDA neurotoxic lesions of the SN pars compacta (SNc) and submitted the rats to a custom-built ASR measurement session to assess amplitude and latency of motor responses. We found that following lesion of the SNc, ASR amplitude decreased and latency increased compared to those values from the sham-surgery and control groups. The number of dopamine neurons remaining in the SNc after lesion was also estimated using a stereological approach, and it correlated with our behavioral results. Moreover, we employed neural tract-tracing techniques to highlight direct projections from the SN to PnC, and indirect projections through the PPTg. Finally, we also measured levels of excitatory amino acid neurotransmitters in the PnC following lesion of the SN, and found that they change following an ipsi/contralateral pattern. Taken together, our results identify nigrofugal efferents onto the primary ASR circuit that may modulate motor responses.
... (corresponding to cholinergic groups Ch5 and Ch6; Armstrong et al. 1983;Mesulam et al. 1983;Satoh et al. 1983), they form a rostrocaudal continuum of cholinergic neurons that extends from the caudal border of the substantia nigra pars reticulata (SNR) to the lateral part of the central gray matter, in the periventricular area near the border of the fourth ventricle Paxinos and Watson 2014). No ultrastructural differences among brainstem cholinergic neurons, regardless of their location, have been identified (Honda and Semba 1995). While there are some clear differences in the density of cholinergic neurons across the rostrocaudal axis (see below), no clear borders or landmarks divide the PPN from the LDT. ...
... Thus, I next describe the functional specialization across the cholinergic brainstem in three main components, rostral PPN (pars rostralis, pr), caudal PPN (pars caudalis, pc) and LDT, and discuss the common features supporting their operation as a single functional entity. The density of cholinergic neurons has been commonly used to delimit the borders of the PPNpr, PPNpc and LDT (Satoh et al. 1983;Rye et al. 1987;Honda and Semba 1995). The most rostral part of the continuum of cholinergic neurons was named the pars dissipata (equivalent to pars rostralis here), because of the sparseness of cholinergic neurons. ...
Cholinergic neurons of the brainstem have traditionally been associated with a role in wakefulness as part of the reticular activating system, but their function cannot be explained solely on the basis of their modulation of the brain state. Recent findings about their connectivity and functional heterogeneity suggest a wider role in behavior, where basal ganglia is at the center of their influence. This review focuses on recent findings that suggest an intrinsic functional organization of the cholinergic brainstem that is closely correlated with its connectivity with midbrain and forebrain circuits. Furthermore, recent evidence on the temporal structure of the activation of brainstem cholinergic neurons reveals fundamental aspects about the nature of cholinergic signaling. Consideration of the cholinergic brainstem complex in the context of wider brain circuits is critical to understand its contribution to normal behavior.
... Along with the most abundant cholinergic neurons, PPN contains also non-cholinergic neurons (15,42,(50)(51)(52). The studies reported by the Bruce Wainer's group (15,52-54) suggested that the term "PPN" should be limited to the cluster of cholinergic neurons that project to the thalamus, whilst the non-cholinergic neurons that are interconnected with the basal ganglia should be separately termed "the midbrain extrapyramidal area". ...
... Neurons in PPN tend to have more irregular shapes than those in LDTN. Somewhat at a difference to the previous report (50), Honda and Semba (51) claimed that the mean number of synapses received by cholinergic somata is greater, by about 70% compared to non-cholinergic perikarya. ...
The present review compiles data on the cytoarchitecture, transmitters, development, afferent and efferent connections of the pedunculopontine tegmental nucleus (PPN). PPN is a reticular formation nucleus, located in the pontomesencephalic tegmentum, closely associated with the ascending limb of the superior cerebellar peduncle. Its most typical cells are cholinergic and comprise the Ch5 neuronal group of Mesulam. It contains also glutamatergic neurons that may contain glutamate as a sole transmitter or as a co-transmitter of acetylcholine. The cholinergic neurons use also the gaseous transmitter nitric oxide, being the most prominent nitrergic neurons in the central nervous system (CNS). In aged animals, there is practically no cell loss but there are certain drastic changes in the somatodendritic morphology. PPN has an extremely rich afferent input. All basal ganglia send axons to PPN, the strongest connection being from the substantia nigra (SN), followed by pathways arising from the subthalamic nucleus (STN) and from both pallidal segments (PAL). PPN receives afferents also from the cerebral cortex, from areas of the limbic system and hypothalamus, from the cerebellum, from the brainstem -particularly serotoninergic axons from the raphe nuclei and noradrenergic axons from the locus ceruleus - as well as from the spinal cord. The efferent connections of PPN are extremely diverse, and some of them are carried out by axons that emit divergent collaterals to two different structures. The heaviest efferent pathway of PPN is destined to the thalamus, innervating virtually all thalamic nuclei, and especially the "nonspecific" intralaminar nuclei, that innervate broad ares of the cerebral cortex. All basal ganglia are innervated and in most cases the connection is bilateral. The most significant pathway innervates the dopaminergic neurons of SN, followed by a connection to STN and PAL. Other PPN efferent connections reach the cerebellum, the superior colliculus, nuclei of cranial nerves, the reticular formation, and the spinal cord. The reviewed connections of PPN suggest that it is involved significantly in the arousal systems, and is implicated in the disturbances of sleep and wakefulness. PPN is also involved in the motor functions of CNS, as well as in the movement disorders.
... Although cholinergic input may also source from outside the LDT, cholinergic LDT neurons themselves are responsible for an ACh-containing input directed to LDT cholinergic cells. Although cholinergic input within the LDT is directed for the most part to noncholinergic LDT neurons, studies of synaptic profiles have suggested that LDT acetylcholine-containing neurons release ACh onto neighboring cholinergic cells within the ipsi-and contralateral LDT [41,57,166]. Inhibitory actions of ACh on cholinergic neurons within the LDT were thought to serve as a feedback mechanism to reduce or terminate ongoing cholinergic activation during periods of LDT-mediated cholinergic activation of the EEG [131,163]. ...
... Instead, the effect might depend on the levels of ACh in the vicinity of particular populations of muscarinic receptors, leading to a signal to noise filtering scenario where small excitatory events in cholinergic neurons would lead to a negative feedback inhibition of cholinergic cells, whereas larger excitatory events would lead to further excitation that may extend to the entire cholinergic network [164]. Dual actions of ACh on postsynaptic cholinergic LDT neurons, coupled with a heavy presence of excitatory, AChcontaining synapses directed to neighboring noncholinergic cells [166], suggest that ACh actions within the LDT would be complex. ...
Drug addiction is a multileveled behavior controlled by interactions among many diverse neuronal groups involving several neurotransmitter systems. The involvement of brainstem-sourced, cholinergic neurotransmission in the development of addiction and in the persistent physiological processes that drive this maladaptive behavior has not been widely investigated. The major cholinergic input to neurons in the midbrain which are instrumental in assessment of reward and assignment of salience to stimuli, including drugs of abuse, sources from acetylcholine- (ACh-) containing pontine neurons of the laterodorsal tegmentum (LDT). Excitatory LDT input, likely cholinergic, is critical in allowing behaviorally relevant neuronal firing patterns within midbrain reward circuitry. Via this control, the LDT is positioned to be importantly involved in development of compulsive, addictive patterns of behavior. The goal of this review is to present the anatomical, physiological, and behavioral evidence suggesting a role of the LDT in the neurobiology underlying addiction to drugs of abuse. Although focus is directed on the evidence supporting a vital participation of the cholinergic neurons of the LDT, data indicating a contribution of noncholinergic LDT neurons to processes underlying addiction are also reviewed. While sparse, available information of actions of drugs of abuse on LDT cells and the output of these neurons as well as their influence on addiction-related behavior are also presented. Taken together, data from studies presented in this review strongly support the position that the LDT is a major player in the neurobiology of drug addiction. Accordingly, the LDT may serve as a future treatment target for efficacious pharmaceutical combat of drug addiction.
... Rhythmical slow activity (RSA) in the hippocampus, described as theta (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), can be recorded during wakefulness, and represents one of the tonic markers of rapid eye movement (REM) sleep. In anesthetized rats, hippocampal theta can be induced by sensory stimulation such as tail pinch, by drug infusion or electrical stimulation of specific brain areas, including structures within the brainstem [4,5,23,24,26,28,37,38,40]. ...
... Serotonin receptors agonists used in this experiment preferentially stimulate the 5-HT 1A receptor subtype but 8-OH-DPAT may also act through 5-HT 7 , and 5-CT, through 5-HT 1B , 5-HT 1D , 5-HT 5 and 5-HT 7 receptor subtypes. In the PPN, serotonin receptors were found on cholinergic, glutamatergic and GABAergic interneurons [12,30]. Steininger et al. [33] showed that in the PPN only 12% of 5-HT axons terminate on cholinergic neurons, and the rest of 5-HT axons, constituting 88%, innervate non-cholinergic, mainly glutaminergic neurons, and hence the dominant influence of serotonin may be exerted on this population of cells. ...
The pedunculopontine tegmental nucleus (PPN), as a part of reticular formation activating system, is thought to be involved in the sleep/wake cycle regulation, and plays an important role in the generation and regulation of hippocampal rhythmical slow activity. The activity of PPN can be modulated by serotonergic system, mainly through multiple projections from raphe nuclei, which can influence PPN neurons through different classes of 5-HT receptors. In the present study, the effect of intra-PPN injection of two serotonin agonists: 8-OH-DPAT and 5-CT, on hippocampal formation EEG activity was examined in urethane-anesthetized rats. The study found that the microinjections induced prolonged spontaneous theta rhythm in both hippocampi with a short latency. The results obtained suggest that local inhibition of presumably cholinergic neurons in the PPN acts as a trigger for hippocampal theta activity.
... Terminals from the laterodorsal tegmental nucleus are characterized by pleomorphic vesicles and make symmetric synaptic connections with ML neurons, presumably constituting an inhibitory input (Hayakawa and Zyo 1992). The laterodorsal tegmental nucleus is well known for the vast presence of cholinergic neurons (Honda and Semba 1995), and is also the main source of cholinergic innervation in the anterior thalamus Hallanger et al. 1987;Holmstrand and Sesack 2011). Interestingly, a significant number of muscarinic receptors in the ATN are presynaptic, and it has been suggested that cholinergic innervation of the ATN may provide a classical presynaptic inhibition through these receptors during the activation of projections from the MB (Sikes and Vogt 1987). ...
The mammillary body (MB) is a component of the extended hippocampal system and many studies have shown that its functions are vital for mnemonic processes. Together with other subcortical structures, such as the anterior thalamic nuclei and tegmental nuclei of Gudden, the MB plays a crucial role in the processing of spatial and working memory, as well as navigation in rats. The aim of this paper is to review the distribution of various substances in the MB of the rat, with a description of their possible physiological roles. The following groups of substances are reviewed: (1) classical neurotransmitters (glutamate and other excitatory transmitters, gamma-aminobutyric acid, acetylcholine, serotonin, and dopamine), (2) neuropeptides (enkephalins, substance P, cocaine- and amphetamine-regulated transcript, neurotensin, neuropeptide Y, somatostatin, orexins, and galanin), and (3) other substances (calcium-binding proteins and calcium sensor proteins). This detailed description of the chemical parcellation may facilitate a better understanding of the MB functions and its complex relations with other structures of the extended hippocampal system.
... In the septal complex of the BF, the dendrites, axon, and soma of cholinergic neurons are mostly surrounded by astrocytic processes (Milner, 1991). In the laterodorsal and pedunculopontine tegmental nuclei of the brainstem, cholinergic neurons receive a large amount of synaptic input, with approximately one-quarter of their somatic surface covered by astrocytic processes (Honda and Semba, 1995). Expression levels of α7nAChR have been investigated in various inflammatory models. ...
Aging is a complex biological process that increases the risk of age-related cognitive degenerative diseases such as dementia, including Alzheimer’s disease (AD), Lewy Body Dementia (LBD), and mild cognitive impairment (MCI). Even non-pathological aging of the brain can involve chronic oxidative and inflammatory stress, which disrupts the communication and balance between the brain and the immune system. There has been an increasingly strong connection found between chronic neuroinflammation and impaired memory, especially in AD. While microglia and astrocytes, the resident immune cells of the central nervous system (CNS), exerting beneficial effects during the acute inflammatory phase, during chronic neuroinflammation they can become more detrimental. Central cholinergic circuits are involved in maintaining normal cognitive function and regulating signaling within the entire cerebral cortex. While neuronal-glial cholinergic signaling is anti-inflammatory and anti-oxidative, central cholinergic neuronal degeneration is implicated in impaired learning, memory sleep regulation, and attention. Although there is evidence of cholinergic involvement in memory, fewer studies have linked the cholinergic anti-inflammatory and anti-oxidant pathways to memory processes during development, normal aging, and disease states. This review will summarize the current knowledge of cholinergic effects on microglia and astroglia, and their role in both anti-inflammatory and anti-oxidant mechanisms, concerning normal aging and chronic neuroinflammation.
... In addition to cholinergic neurons, the PPN is populated by glutamatergic and GABAergic neurons (Clements and Grant, 1990;Clements et al., 1991;Ford et al., 1995;Lavoie and Parent, 1994b). Cholinergic neurons, which express choline acetyltransferase (ChAT) and NADPH (Clements and Grant, 1990), have medium to large fusiform, triangular, multipolar or round somas (20-40 μm in diameter) with 2 to 6 primary dendrites (Honda and Semba, 1995;Ichinohe et al., 2000;Rye et al., 1987). Glutamatergic neurons, which express type 2 vesicular glutamate transporter (VGlut2), have a smaller somata (< 20 μm) with 2 to 4 primary dendrites (Clements and Grant, 1990;Ichinohe et al., 2000;Jia et al., 2003;Wang and Morales, 2009). ...
In the last decade, scientific and clinical interest in the pedunculopontine nucleus (PPN) has grown dramatically. This growth is largely a consequence of experimental work demonstrating its connection to the control of gait and of clinical work implicating PPN pathology in levodopa-insensitive gait symptoms of Parkinson's disease (PD). In addition, the development of optogenetic and chemogenetic approaches has made experimental analysis of PPN circuitry and function more tractable. In this brief review, recent findings in the field linking PPN to the basal ganglia and PD are summarized; in addition, an attempt is made to identify key gaps in our understanding and challenges this field faces in moving forward.
... Nevertheless, more recently, Alderson et al. (2008) have observed a reduction in locomotion after lesioning a restricted portion of the anterior but not of the posterior part of the PPTg. These results are consistent with the hypothesis that in rats the anterior PPTg (aPPTg), which is thought to resemble the PPNd, has functions and anatomical connections related to motor processes (Rye et al., 1987;Honda and Semba, 1995), while the posterior PPTg (pPPTg) resembles the PPNc because of a high density of cholinergic neurons and stronger anatomical connections to associativelimbic structures (Olszewski and Baxter, 1982;Manaye et al., 1999;Mena-Segovia et al., 2008). Whether the effects of the aPPTg lesions are achieved through the effects on descending motor projections, or through effects on the BG motor loop, possibly via the CnF as suggested by Alam et al. (2012), has not been investigated. ...
Loss of cholinergic neurons in the mesencephalic locomotor region, comprising the pedunculopontine nucleus (PPN) and the cuneiform nucleus (CnF), are related to gait disturbances in late stage Parkinson's disease (PD). We investigate the effect of anterior or posterior cholinergic lesions of the PPN on gait-related motor behaviour, and on neuronal network activity of the PPN area and basal ganglia (BG) motor loop in rats. Anterior PPN lesions, posterior PPN lesions or sham lesions were induced by stereotaxic microinjection of the cholinergic toxin AF64-A or vehicle in male Sprague Dawley rats. First, locomotor activity (open field), postural disturbances (Rotarod) and gait asymmetry (treadmill test) were assessed. Thereafter, single unit and oscillatory activity were measured in the non-lesioned area of the PPN, the CnF and the entopeduncular nucleus (EPN), the BG output region, with microelectrodes under urethane anaesthesia. Additionally, ECoG was recorded in the motor cortex. Injection of AF64-A into the anterior and posterior PPN decreased cholinergic cell counts as compared to naive controls (P<0.001) but also destroyed non-cholinergic cells. Only anterior PPN lesions decreased the front limb swing time of gait in the treadmill test, while not affecting other gait related parameters tested. Main electrophysiological findings were that anterior PPN lesions increased the firing activity in the CnF (P<0.001). Further, lesions of either PPN region decreased the coherence of alpha (8-12Hz) band between CnF and MCx, and increased the beta (12-30Hz) oscillatory synchronization between EPN and the MCx. Lesions of the PPN in rats had complex effects on oscillatory neuronal activity of the CnF and the BG network, which may contribute to the understanding of the pathophysiology of gait disturbance in PD.
... In the VTA to NAc circuit, DA burst firing and phasic DA release is mediated by N-methyl-D-aspartate receptor (NMDAR) and acetylcholine receptor (AChR) mechanisms Blaha, 2000, 2003;Grace et al., 2007;Sombers et al., 2009;Wickham et al., 2013) and recent evidence points toward a role for VTA NMDARs and AChRs in cue-dependent behavior. In particular, the mesopontine tegmentum (MPT), which contains the laterodorsal tegmentum (LDTg) and pedunculopontine tegmentum (PPTg), sends both cholinergic and glutamatergic projections to the VTA (Clements et al., 1991;Honda and Semba, 1995;Oakman et al., 1995;Takakusaki et al., 1996). PPTg inactivation has been shown to impair stimulus-reward learning, conditioned reinforcement (Inglis et al., 2000) and also impairs the ability of VTA DA neurons to burst fire in the presence of reward-predictive cues (Pan et al., 2005). ...
Stimuli paired with rewards acquire reinforcing properties to promote reward-seeking behavior. Previous work supports the role of ventral tegmental area (VTA) nicotinic acetylcholine receptors (nAChRs) in mediating conditioned reinforcement elicited by drug-associated cues. However, it is not known whether these cholinergic mechanisms are specific to drug-associated cues or whether VTA cholinergic mechanisms also underlie the ability of cues paired with natural rewards to act as conditioned reinforcers. Burst firing of VTA dopamine (DA) neurons and the subsequent phasic DA release in the nucleus accumbens (NAc) plays an important role in cue-mediated behavior and in the ability of cues to acquire reinforcing properties. In the VTA, both AChRs and N-methyl-d-aspartate receptors (NMDARs) regulate DA burst firing and phasic DA release. Here, we tested the role of VTA nAChRs, muscarinic AChRs (mAChRs), and NMDARs in the conditioned reinforcement elicited by a food-associated, natural reward cue. Subjects received 10 consecutive days of Pavlovian conditioning training where lever extension served as a predictive cue for food availability. On day 11, rats received bilateral VTA infusion of saline, AP-5 (0.1 or 1 μg), mecamylamine (MEC: 3 or 30 μg) or scopolamine (SCOP: 3 or 66.7 μg) immediately prior to the conditioned reinforcement test. During the test, nosepoking into the active (conditioned reinforced, CR) noseport produced a lever cue while nosepoking on the inactive (non-conditioned reinforced, NCR) noseport had no consequence. AP-5 robustly attenuated conditioned reinforcement and blocked discrimination between CR and NCR noseports at the 1-μg dose. MEC infusion decreased responding for both CR and NCR while 66.7-μg SCOP disrupted the subject’s ability to discriminate between CR and NCR. Together, our data suggest that VTA NMDARs and mAChRs, but not nAChRs, play a role in the ability of natural reward-associated cues to act as conditioned reinforcers.
... Studies assessing the complex anatomic connectivity of the PPN, as well as the functional implications of this connectivity, have spanned both multiple modalities and species. Cytoarchitectural/immunohistochemical [14][15][16][17][18][19] and anterograde/ retrograde tracing studies [20][21][22] have allowed identification of anatomically and neurochemically distinct ascending/ descending inputs/outputs as well as distinct cholinergic and dopaminergic neuronal subpopulations that are shared between species [23,24]. Further, recent diffusion tensor imaging tractography studies have shown significant homology between the functional connectivity of the PPN in primates and lower mammals [25]. ...
Deep brain stimulation has proven an effective addition to the optimized medical management of some primary movement disorders. Sustained symptomatic improvement has been demonstrated for Parkinson's disease, forms of dystonia, and essential tremor. However, despite dramatic improvements in the tremor, rigidity, bradykinesia, and dyskinesias of Parkinson's disease, DBS of the STN and GPi have provided inconsistent relief of gate abnormalities and freezing. Similarly, DBS of the Vim has proven effective for distal tremors resulting from a variety of etiologies, but has limited efficacy for tremors with proximal spread. Accumulating clinical, neurophysiologic, and neuroanatomic evidence supports the pedunculopontine nucleus as a modulator of postural control and gait initiation. Further, both historical and contemporary preclinical and clinical data support the zona incerta and prelemniscal radiations as targets within the greater subthalamic area for tremor containing proximal spread. On the basis of these observations, there is considerable interest in PPN DBS for control of gate abnormalities in PD, and in ZI and PRL DBS as a means for modulation of pronounced tremor with axial involvement. The clinical evidence for consideration of the PPN and ZI/PRL as alternate stimulation targets for treatment of refractory movement disorder manifestations is reviewed.
... Further, we demonstrated the presence of a local glutamate circuit within the LDT which exhibited ongoing activity in the brain slice (Kohlmeier et al. 2012). Taken together, these data suggest that the endogenous release of glutamate in the LDT from local neurons or terminals of projection neurons (Honda and Semba 1995) could induce DSI via actions on mGluRs on cells in this nucleus. In our earlier study, recording conditions were not optimized to detect whether the application of mGluR1 agonists affected the frequency or amplitude of sIPSCs that we have shown are mediated in LDT cells by GABAergic mechanisms (Ishibashi et al. 2009). ...
Cannabinoid type 1 receptors (CB1Rs) are functionally active within the laterodorsal tegmental nucleus (LDT), which is critically involved in control of rapid eye movement sleep, cortical arousal, and motivated states. To further characterize the cellular consequences of activation of CB1Rs in this nucleus, we examined whether CB1R activation led to rises in intracellular Ca2+ ([Ca2+]i) and whether processes shown in other regions to involve endocannabinoid (eCB) transmission were present in the LDT. Using a combination of Ca2+ imaging in multiple cells loaded with Ca2+ imaging dye via ‘bulk-loading’ or in single cells loaded with dye via a patch-clamp electrode, we found that WIN 55212-2 (WIN-2), a potent CB1R agonist, induced increases in [Ca2+]i which were sensitive to AM251, a CB1R antagonist. A proportion of rises persisted in TTX and/or low-extracellular Ca2+ conditions. Attenuation of these increases by a reversible inhibitor of sarcoplasmic reticulum Ca2+-ATPases, suggests these rises occurred following release of Ca2+ from intracellular stores. Under voltage clamp conditions, brief, direct depolarization of LDT neurons resulted in a decrease in the frequency and amplitude of AM251-sensitive, inhibitory postsynaptic currents (IPSCs), which was an action sensitive to presence of a Ca2+ chelator. Finally, actions of DHPG, a mGlu1R agonist, on IPSC activity were examined and found to result in an AM251- and BAPTA-sensitive inhibition of both the frequency and amplitude of sIPSCs. Taken together, our data further characterize CB1R and eCB actions in the LDT and indicate that eCB transmission could play a role in the processes governed by this nucleus.
... The cholinergic neurons have larger somata, but are less numerous than the noncholinergic neurons (Honda and Semba, 1995;Rye et al., 1987;Spann and Grofova, 1992). ...
... Both the PPT and the LDT contain cholinergic (Armstrong et al., 1983;Clarke and Kumar, 1983a;Sugimoto and Hattori, 1984;Beninato and Spencer, 1987;Beninato and Spencer, 1988;Gould et al., 1989;Bolam et al., 1991;Butcher et al., 1992) and noncholinergic (Spann and Grofova, 1992;Honda and Semba, 1995;Steininger et al., 1997;Wang and Morales, 2009) neurons. The non-cholinergic neurons (e.g. ...
... Several lines of evidence support the reciprocal interaction model. The supporting evidence includes but is not limited to the following: (i) exogenous cholinergic stimulation of the SLDn and other region in the PRF induces REM sleep (see sec.1.4.1), (ii) microdialysis studies show that endogenous levels of acetylcholine in the PRF increase during REM sleep (Kodama et al., 1990;Lydic et al., 1991), (iii) The SLDn is innervated by cholinergic LDTn and PPTn neurons (Datta et al., 1999), (iv) the LDTn and PPTn contain neuronal sub-groups exhibiting maximal activity during REM sleep (Thakkar et al., 1998), (v) Electrical stimulation of the LDTn evokes excitatory post synaptic potentials in PRF neurons that can be blocked by scopolamine , (vi) electrical stimulation of the LDTn promotes REM sleep (Thakkar et al., 1996), (vii) serotonergic neurons of the DRN have been shown to project to the LDTn and PPTn (Honda and Semba, 1995), and (viii) serotonin type 1A receptor agonism selectively inhibits activity of REM sleep-active PPTn and LDTn neurons (Thakkar et al., 1998 The PPTn was first defined on cytoarchitectonic grounds in the human brain. It consists of a collection of large neurons that extend from the caudal pole of the red nucleus to the parabrachial nucleus in close association with the ascending limb of the superior cerebellar peduncle (Rye et al., 1987). ...
... these results have suggested that the excitatory amino acids, at least partially, Sato and Fibiger (1986 could activate tegmental cholinergic neurons with the ascending pathways and induce defensive responses in this way. the main group of cholinergic cell bodies is localized in the laterodorsal tegmental nucleus (Ch6 group), pedunculopontine nucleus (Ch5 group) and some neighboring structures, including the ventral periaqueductal gray (honda and Semba, 1995;Motts et al., 2008;Wang and Morales, 2009). however, the main ascending projections to the diencephalic and forebrain regions originate in the laterodorsal tegmental nucleus (Satoh and Fibiger, 1986;hallanger and Wainer, 1988;Cornwall et al., 1990;Woolf et al., 1990) (see Fig. 4). ...
AbstractPharmacological studies performed on cat and rat brains are reviewed, which have allowed for identification of a widespread cholinoceptive system in the mammalian brain responsible for initiation of defensive vocalizations characteristic of aversive behavioral situations. Intracerebral injections of a predominantly muscarinic agent, carbachol, induced growling and hissing vocalizations in cats and 22 kHz ultrasonic alarm vocalizations in rats. Brain systems inducing these calls and their neurochemical organization in both the species show a very high degree of homology. This cholinoceptive substrate for these vocalizations, termed the medial cholinoceptive vocalization strip, is innervated by the ascending fibers from the brainstem cholinergic neurons located in the laterodorsal tegmental nucleus. This nucleus forms a cholinergic component of the ascending activating reticular system and its functions are discussed.
... However, it was shown that PPT dendrites extend primarily into adjacent sensory pathways (Rye et al., 1987;Semba, 1991). Additionally, it is possible, that glutamate receptors of PPT neurons are heavily located on their soma (Honda and Semba, 1995). ...
Functionally distinct areas were mapped within the pedunculopontine tegmentum (PPT) of 42 ketamine/xylazine anesthetized rats using local stimulation by glutamate microinjection (10 mM, 5-12 nl). Functional responses were classified as: (1) apnea; (2) tachypnea; (3) hypertension (HTN); (4) sinus tachycardia; (5) genioglossus electromyogram activation or (6) pontine-waves (p-waves) activation.We found that short latency apneas were predominantly elicited by stimulation in the lateral portion of the PPT, in close proximity to cholinergic neurons. Tachypneic responses were elicited from ventral regions of the PPT and HTN predominated in the ventral portion of the antero-medial PPT. We observed sinus tachycardia after stimulation of the most ventral part of the medial PPT at the boundary with nucleus reticularis pontis oralis, whereas p-waves were registered predominantly following stimulation in the dorso-caudal portion of the PPT. Genioglossus EMG activation was evoked from the medial PPT. Our results support the existence of the functionally distinct areas within the PPT affecting respiration, cardiovascular function, EEG and genioglossus EMG.
... These data indicate that some VTA neurons receive only glutamatergic inputs from the PPN, rather than a mix of GABAergic and glutamatergic inputs. This is consistent with the identification of three major independent projection neurons in the PPN that use GABA or glutamate (Sugimoto & Hattori, 1984; Clements & Grant, 1990; Ford et al. 1995), or ACh, or ACh co-localized with glutamate (Clements et al. 1991; Lavoie & Parent, 1994; Honda & Semba, 1995). However, a more recent study has demonstrated that greater than 95% of all neurons in the PPN and LDT expressing the cholinergic marker choline acetyltransferase (ChAT) did not express markers for GABAergic (glutamic acid decarboxylase mRNA) or glutamatergic (type-2 vesicular glutamate transporter, vGluT2 mRNA) neurons, strongly suggesting the existence of three wholly independent populations of projection neurons in these nuclei (Wang & Morales 2009). ...
Anatomical studies indicate that synaptic inputs from many cortical and subcortical structures converge on neurons of the ventral tegmental area (VTA). Although in vitro electrophysiological studies have examined synaptic inputs to dopamine (DA) and non-DA neurons in the VTA, they have largely relied upon local electrical stimulation to activate these synapses. This provides little information regarding the distinct properties of synapses originating from different brain areas. Using whole-cell recordings in parasagittal rat brain slices that preserved subcortical axons from the pedunculopontine nucleus (PPN) to the VTA, we compared these synapses with those activated by intra-VTA stimulation. PPN-evoked currents demonstrated longer latencies than intra-VTA-evoked currents, and both VTA and PPN responses were mediated by GABA(A) and AMPA receptors. However, unlike VTA-evoked currents, PPN currents were exclusively mediated by glutamate in 25-40% of the VTA neurons. Consistent with a cholinergic projection from the PPN to the VTA, nicotinic acetylcholine receptors (nAChR) were activated by endogenous acetylcholine released during PPN, but not VTA, stimulation. This was seen as a reduction of PPN-evoked, and not VTA-evoked, synaptic currents by the alpha7-nAChR antagonist methyllycaconitine (MLA) and the agonist nicotine. The beta2-nAChR subunit antagonist dihydro-beta-erythroidine had no effect on VTA- or PPN-evoked synaptic currents. The effects of MLA on PPN-evoked currents were unchanged by the GABA(A) receptor blocker picrotoxin, indicating that alpha7-nAChRs presynaptically modulated glutamate and not GABA release. These differences in physiological and pharmacological properties demonstrate that ascending PPN and presumed descending inputs to VTA utilize distinct mechanisms to differentially modulate neuronal activity and encode cortical and subcortical information.
Stress is a physiological response that promotes maintenance of balance against harmful stimuli. Unfortunately, chronic activation of stress systems facilitates the development of psychiatric disorders. A stress-mediated hypercholinergic state could underlie this facilitation, as cholinergic mechanisms have been suggested to play a role in anxiety, depression, and substance use disorder (SUD). Stimulation by stress hormones, urocortin (Ucn1) or corticotropin-releasing factor (CRF), of the CRF receptor type 1 (CRFR1) of acetylcholine-containing neurons of the laterodorsal tegmental nucleus (LDT) could be involved in modulation of cholinergic transmission during periods of stress hormone activation, which could play a role in psychiatric disorders as cholinergic LDT neurons project to, and control activity in, mood-, arousal- and SUD-controlling regions. The present study investigated for the first time the membrane effects and intracellular outcomes of CRFR1 activation by endogenous stress hormones on LDT neurons. Patch clamp recordings of immunohistochemically-identified cholinergic and non-cholinergic LDT neurons with concurrent calcium imaging were used to monitor cellular responses to CRFR1 stimulation with Ucn1 and CRF. Postsynaptically-mediated excitatory currents were elicited in LDT cholinergic neurons, accompanied by an enhancement in synaptic events. In addition, CRFR1 activation resulted in rises in intracellular calcium levels. CRFR1 stimulation recruited MAPK/ERK and SERCA-ATPase involved pathways. The data presented here provide the first evidence that Ucn1 and CRF exert pre and postsynaptic excitatory membrane actions on LDT cholinergic neurons that could underlie the hypercholinergic state associated with stress which could play a role in the heightened risk of psychiatric disorders associated with a chronic stress state.
Electrical stimulation of the anterior pretectal nucleus (APtN) activates two descending pain inhibitory pathways. One of these pathways relays in the ipsilateral lateral paragigantocellular nucleus (LPGi), whereas the other pathway relays in the contralateral pedunculopontine tegmental nucleus (PPTg). Antinociceptive effect of APtN stimulation has been seen in various pain models in the rodents. Similarly, LPGi or PPTg stimulation results in higher pain thresholds. Descending antinociceptive pathways activated by electrical APtN stimulation have been elucidated, but the underlying neurotransmitter mechanisms involved have not been clarified yet. This study investigates the role that endogenous signaling plays in the ipsilateral LPGi or contralateral PPTg after the APtN is stimulated in the tail-flick test. First, we submitted rats to excitotoxic injection of N-methyl-D-aspartate (NMDA) into the contralateral PPTg. Then, we examined whether blockage of NMDA (AP-7), serotonergic (methysergide), or opioid (naloxone) receptors in the ipsilateral LPGi is required for APtN stimulation-evoked analgesia (SEA). Likewise, we examined the effects of antagonists of NMDA, serotonergic, or cholinergic nicotinic (mecamylamine) receptors on the contralateral PPTg in ipsilateral LPGi-lesioned rats. Our results confirmed that APtN stimulation activates two pain inhibitory pathways and showed that endogenous opioid signaling in the ipsilateral LPGi appears to be necessary for APtN SEA and for endogenous NMDA, serotoninergic, and nicotinergic signaling in the contralateral PPTg.
The pedunculopontine nucleus (PPN) is a reticular nucleus located in the mesencephalic and upper pontine tegmentum. Initially, characterized by its predominant cholinergic projection neurons, it was associated with the "mesencephalic locomotor region" and "reticular activating system". Furthermore, based on histopathological studies, the PPN was hypothesized to play a role in the manifestation of symptoms in movement disorders such as Parkinson's disease (PD). Since axial symptoms represent unmet needs of PD treatments, a series of pioneering experiments in Parkinsonian monkeys promoted the idea of a potential new target for deep brain stimulation (DBS) and much clinical interest was generated in the following years leading to a number of trials analysing the role of PPN for gait disorders. This review summarizes the historical background and more recent findings about the anatomy and function of the PPN and its implications in the basal ganglia network of the normal as well as diseased brain. Classical views on PPN function shall be challenged by more recent findings. Additionally, the current role and future perspectives of PPN DBS in PD patients shall be outlined.
Anatomical studies have revealed dense connections between the pedunculopontine tegmental nucleus (PPN) and the basal ganglia (Figure 1). In primates, PPN receives massive afferents from the two output structures of the basal ganglia, that is, the substantia nigra pars reticulata and the internal pallidum. In turn, PPN projects profusely to the substantia nigra pars compacta and the subthalamic nucleus (Filion and Harnois, 1978; Harnois and Filion, 1980; Harnois and Filion, 1982; Parent and Hazrati, 1995). These ascending projections were found both ipsilaterally and contralaterally (Lavoie and Parent, 1994b). The existence of cerebello-tegmental projections, which are ipsilateral collaterals of the cerebello-thalamic projection, was also reported (Hazrati and Parent, 1992). Motor cortex (area 4) also sends fibers to PPN (Hartmann-Von Monakow et al., 1979; Moon Edley and Graybiel, 1983). PPN is a part of the mesencephalic locomotor region, from which locomotor movements are induced by its electrical stimulation (Garcia-Rill, 1986; Garcia-Rill, 1991).
Background:
Identification of cell phenotype from brain slices upon which in vitro electrophysiological recordings have been performed often relies on conducting post hoc immunohistochemistry on tissue that necessarily has not been ideally prepared for immunohistochemical procedures. In such studies, antibody labeling against neuronal nitric oxide synthase (bNOS) has been used to identify cholinergic neurons of the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nuclei (PPT), two brainstem nuclei importantly involved in arousal. However, a widespread perception maintains that antibody staining for enzymes involved in synthesis or transport, of acetylcholine would be a more definitive marker and hence, preferable.
New method:
Colocalization of bNOS and CHAT in the LDT/PPT, and presence of parvalbumin (PV), was examined in non-ideally prepared mouse brain slices using currently available antibodies.
Results:
Using fluorescent-based immunohistochemistry in LDT/PPT slices prepared for in vitro recordings, a near 100% colocalization of bNOS and CHAT was observed.
Comparison with existing method:
We confirm in the mouse, findings of near 100% colocalization of bNOS and CHAT in the LDT/PPT, and we expand upon data from rat studies using optimally prepared tissue, that for dendritic visualization, bNOS staining exceeded the quality of CHAT staining for visualization of a higher degree of detail of fine processes. PV is not highly present in the mouse LDT/PPT.
Conclusion:
CHAT and bNOS are equally useful target proteins for immunofluorescent identification of cholinergic LDT/PPT cells in mouse brain slices prepared for in vitro recordings, however, antibody targeting of bNOS allows for a superior appreciation of structural detail.
Ghrelin, a gut and brain peptide, has recently been shown to be involved in motivated behavior and regulation of the sleep and wakefulness cycle. The laterodorsal tegmental nucleus (LDT) is involved in appetitive behavior and control of the arousal state of an organism, and accordingly, behavioral actions of ghrelin could be mediated by direct cellular actions within this nucleus. Consistent with this interpretation, postsynaptically mediated depolarizing membrane actions of ghrelin on LDT neurons have been reported. Direct actions were ascribed solely to closure of a potassium conductance however this peptide has been shown in other cell types to lead to rises in calcium via release of calcium from intracellular stores. To determine whether ghrelin induced intracellular calcium rises in mouse LDT neurons, we conducted calcium imaging studies in LDT brain slices loaded with the calcium binding dye, Fura-2AM. Ghrelin elicited TTX-insensitive changes in dF/F indicative of rises in calcium, and a portion of these rises were independent of membrane depolarization, as they persisted in conditions of high extracellular potassium solutions and were found to involve SERCA-pump mediated intracellular calcium stores. Involvement of the ghrelin receptor (GHR-S) in these actions was confirmed. Taken together with other studies, our data suggest that ghrelin has multiple cellular actions on LDT cells. Ghrelin's induction of calcium via intracellular release in the LDT could play a role in behavioral actions of this peptide as the LDT governs processes involved in stimulation of motivated behavior and control of cortical arousal.
Abstract The subthalamic nucleus (STN) is a key component of the basal ganglia. As the only basal ganglia nucleus comprised of mostly glutamatergic neurons, STN neurons provide a key driving force to their target neurons. Thus, regulation of STN neuron activity is important. One STN regulator is the serotonin (5-HT) system. The STN receives a dense 5-HT innervation. 5-HT1A, 5-HT1B, 5-HT2C, and 5-HT4 receptors are expressed in the STN. 5-HT may regulate the STN via several mechanisms. First, 5-HT may affect STN neuron excitability directly by either inhibiting a subpopulation of STN neurons via activation of 5-HT1A receptors or exciting STN neurons through activation of 5-HT2C and 5-HT4 receptors. Second, 5-HT may affect synaptic inputs to the STN. Via activation of 5-HT1B receptors on the afferent terminals, 5-HT inhibits glutamatergic input to the STN, but the inhibitory effect on GABAergic input is smaller. Third, 5-HT may regulate the STN glutamatergic output by activating presynaptic 5-HT1B receptors, thus reducing burst firing in target neurons. Last, 5-HT may affect glutamate release at the intra-STN axon collaterals and regulate the recurrent excitation. These mechanisms may work in concert to fine-tune the intensity and pattern of STN activity and reduce STN output bursts.
This chapter focuses on the diffuse, ascending cholinergic projection systems, the basal forebrain, and the mesopontine large-celled cholinergic cell groups. In the Nissl-stained sections, the basal forebrain contains a more or less continuous band of magnocellular neurons that begins rostrally at the level of the septum in a medial position and extends caudally and laterally near the anterior commissure. Along its extent, these magnocellular neurons are found either clustered or scattered. The septum in primates contains relatively few neurons (compared to rodents). In the Nissl-stained sections, the parenchyma consists mainly of glial cells, among which are scattered neurons. The medial septal nucleus (MS) is an ill-defined cluster of magnocellular neurons, along the pial border of the structure. The MS disappears at the level of the descent of the columns of the fornix, which coincides approximately with the decussation of the anterior commissure. At this level, the nucleus of the diagonal band of Broca (nDBB) forms a cluster of neurons in the angle between the mid-line and the olfactory tubercle.
Because our knowledge of cholinergic systems in the brains of amphibians is limited, the present study aimed to provide detailed information on the distribution of cholinergic cell bodies and fibers as revealed by immunohistochemistry with antibodies directed against the enzyme choline acetyltransferase (ChAT). To determine general and derived features of the cholinergic systems within the class of Amphibia, both anuran (Rana perezi, Xenopus laevis) and urodele (Pleurodeles waltl) amphibians were studied. Distinct groups of ChAT-immunoreactive cell bodies were observed in the basal telencephalon, hypothalamus, habenula, isthmic nucleus, isthmic reticular formation, cranial nerve motor nuclei, and spinal cord. Prominent plexuses of cholinergic fibers were found in the olfactory bulb, pallium, basal telencephalon, ventral thalamus, tectum, and nucleus interpeduncularis. Comparison of these results with those obtained in other vertebrates, including a segmental approach to correlate cell populations, reveals that the cholinergic systems in amphibians share many features with amniotes. Thus, cholinergic pedunculopontine and laterodorsal tegmental nuclei could be identified in the amphibian brain. The finding of weakly immunoreactive cells in the striatum of Rana, which is in contrast with the condition found in Xenopus, Pleurodeles, and other anamniotes studied so far, has revived the notion that basal ganglia organization is more preserved during evolution than previously thought. J. Comp. Neurol. 382:499-534, 1997. © 1997 Wiley-Liss Inc.
The notion of the pedunculopontine nucleus (PPN) is being updated in light of increasing evidence that identifies it as a
key structure in the processing of exteroceptive information and its transmission to the forebrain, influencing the activity
of PPN’s multiple targets (from basal ganglia to the cerebral cortex). Here we review the anatomical evidence supporting the
existence of a local network that forms the basis for such processing functions and propose a more complex relationship with
its efferent systems. We also identify some of the critical issues that remain to be answered in order to have a comprehensive
understanding of the roles of the PPN. A better insight into this connectivity will also help to understand the contribution
of the PPN to the diverse systems it influences.
The REM phase of sleep has long been of interest because of its association with dreaming and its presence in almost all mammals. We are now beginning to understand the mechanisms of its rhythmic generation, and review current hypotheses in this article. A group of cholinergic neurons at the junction of the pons and midbrain, in the laterodorsal and pedunculopontine tegmental nuclei, begins to discharge before the onset of this phase of sleep. Projections to key brain stem reticular formation regions lead, primarily through actions of non-M1 muscarinic receptors, to heightened excitability and discharge activity in these effector regions for the phenomena of REM sleep. Cholinergic projections to the thalamus promote EEG activation. These mesopontine cholinergic neurons are, in turn, modulated by inhibitory and REM-suppressive projections: norepinephrinergic locus coeruleus projections act as α2and serotonergic dorsal raphe projections act as 5-HT1Areceptors. These mesopontine cholinergic neurons are self-modulating through recurrent collaterals and projections between different subgroups that act as muscarinic and nicotinic receptors. In addition, metabolically generated adenosine acts to inhibit these cholinergic neurons. All of the preceding inhibitory effects are mediated by inwardly rectifying potassium currents. Implications of this neural network for a model of REM sleep cycle generation are discussed.
Ghrelin is a potent stimulant for growth hormone (GH) secretion and feeding. Recent studies further show a critical role of ghrelin in the regulation of sleep-wakefulness. Pedunculopontine tegmental nucleus (PPT), which regulates waking and rapid eye movement (REM) sleep, expresses GH secretagogue receptors (GHS-Rs). Thus, the present study was carried out to examine electrophysiological effects of ghrelin on PPT neurons using rat brainstem slices, and to determine the ionic mechanism involved. Whole cell recording revealed that ghrelin depolarizes PPT neurons dose-dependently in normal artificial cerebrospinal fluid (ACSF). The depolarization persisted in tetrodotoxin-containing ACSF, although action potentials did not occur. Application of [d-Lys3]-GHRP-6, a selective antagonist for GHS-Rs, almost blocked the ghrelin-induced depolarization. Furthermore, the ghrelin-induced depolarization was reduced in high K+ ACSF or low Na+ ACSF, and abolished in high K+–low Na+ ACSF or in a combination of low Na+ ACSF and recordings with Cs+-containing pipettes. An inhibitor of Na+/Ca2+ exchanger had no effect on the depolarization. Most of the PPT neurons recorded were characterized by an A-current or both the A-current and a low threshold Ca2+ spike, and they were predominantly cholinergic as revealed by nicotinamide adenine dinucleotide phosphate-diaphorase staining. These results suggest that ghrelin depolarizes PPT neurons postsynaptically and dose-dependently via GHS-Rs, and that the ionic mechanisms underlying the ghrelin-induced depolarization include a decrease of K+ conductance and an increase of non-selective cationic conductance. The results also support the notion that ghrelin plays a role in the regulation of sleep-wakefulness.
Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) are importantly involved in neurobiological mechanisms governing states of arousal such as sleep and wakefulness as well as other appetitive behaviors, such as drug-seeking. Accordingly, mechanisms controlling their excitability are important to elucidate if we are to understand how these LDT neurons generate arousal states. Glutamate mediates the vast majority of excitatory synaptic transmission in the vertebrate CNS and while presence of glutamate input in the LDT has been shown and ionotropic responses to glutamate have been reported in the LDT, characterization of metabotropic responses is lacking. Therefore, electrophysiological responses and changes in levels of intracellular Ca(2+) in mouse cholinergic LDT neurons following application of specific mGluR agonists and antagonists were examined. Unexpectedly, both the mGluR(5)specific agonist, CHPG, and the group II mGluR (mGlu(2/3)) agonist, LY379268 (LY), induced a TTX-insensitive outward current/hyperpolarization. Both outward currents were significantly reduced by the mGluR antagonist MCPG and the CHPG-induced current was blocked by the specific mGluR(5) antagonist MTEP. Concurrent Ca(2+)imaging revealed that while CHPG actions did include release of Ca(2+) from CPA/thapsigargin-sensitive intracellular stores, actions of LY did not. Both CHPG- and LY-induced outward currents were mediated by a TEA-sensitive potassium conductance. The large-conductance, Ca(2+)-dependent potassium (BK) channel blocker, iberiotoxin, attenuated CHPG actions. Consistent with actions on the BK conductance, CHPG enhanced the amplitude of the fast component of the after hyperpolarizing potential, concurrent with a reduction in the firing rate. We conclude that stimulation of mGluR(5) and group II (mGluR(2/3)) elicits postsynaptically-mediated outward currents/hyperpolarizations in cholinergic LDT neurons. Effects of glutamatergic input would be, thus, expected not only to be excitation via stimulation of ionotropic glutamate receptors and mGluR(1), but also inhibition via actions at mGluR(5) and mGluR(2/3) on these neurons. As these two processes counteract each other, these surprising findings necessitate revision of predictions regarding the net level of excitation generated by glutamate input to cholinergic LDT cells and, by extension, the functional outcome of glutamate transmission on processes which these neurons regulate.
From indirect evidence we have proposed that cholinergic versus non-cholinergic neurons in the laterodorsal tegmental nucleus can be distinguished with the duration of their extracellularly recorded action potentials, "broad" spikes for the former, "brief" for the latter. To test this assumption more directly, we labelled single neurons recorded extracellularly in and around the laterodorsal tegmental nucleus with biocytin or neurobiotin, and processed the sections with reduced nicotinamide adenine dinucleotide phosphate-diaphorase, a proven marker for cholinergic neurons in the laterodorsal tegmental nucleus. Biocytin or neurobiotin which was deposited at the site of recording was incorporated into single neurons. Among 171 trials (91 for broad-spike and 80 for brief-spike neurons), marking was successful in 68 cases (29 for broad-spike and 39 for brief-spike neurons). Almost all (21/22) of the broad-spike neurons located within the laterodorsal tegmental nucleus were positive for reduced nicotinamide adenine dinucleotide phosphate-diaphorase staining, i.e. they were cholinergic, while all of the brief-spike neurons in and outside of the laterodorsal tegmental nucleus lacked the diaphorase activity, and were thus non-cholinergic. The present study shows that, after extracellular labelling of single neurons by biocytin or neurobiotin, cholinergic neurons in the laterodorsal tegmental nucleus are confidently distinguished from non-cholinergic ones in the corresponding area with their spike shapes. It is also shown that the cholinergic neurons distinguished by this criterion are characterized by their tonic firing at slightly lower rate and larger cell size than the brief-spike non-cholinergic ones.
This chapter focuses on the cellular and neurophysiological/neuropharmacological, with most of the emphasis on mechanisms relevant to rapid eye movement (REM) sleep. The chapter presents the sleep architecture and phylogeny/ontogeny so as to provide a basis for the later mechanistic discussions. The chapter discusses the REM sleep and the relevant anatomy and physiology, and describes the role of hypocretin/orexin in REM sleep control. Sleep may be divided into two phases. REM sleep is most often associated with vivid dreaming and a high level of brain activity. The other phase of sleep, called non-REM sleep or slow-wave sleep (SWS), is usually associated with reduced neuronal activity; thought content during this state in humans is, unlike dreams, usually nonvisual and consisting of ruminative thoughts. REM sleep in humans is defined by the presence of low-voltage fast EEG activity, suppression of muscle tone (usually measured in the chin muscles) and the presence of REMs. The REM sleep cycle length is 90 minutes in humans and the duration of each REM sleep episode after the first is approximately 30 minutes. While electroencephalography (EEG) staging of REM sleep in humans usually shows a fairly abrupt transition from non-REM to REM sleep, recording of neuronal activity in animals presents a quite a different picture. Neuronal activity begins to change long before the EEG signs of REM sleep are present.
Adult rats emit 22 kHz ultrasonic alann calls in aversive situations. This type of call IS a component of defensive behaviour and it functions predominantly to warn conspecifics about predators. Production of these calls is dependent on the central cholinergic system. The laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) contain largely cholinergic neurons, which create a continuous column in the brainstem. The LDT projects to structures in the forebrain, and it has been implicated in the initiation of 22 kHz alarm calls. It was hypothesized that release of acetylcholine from the ascending LDT terminals in mesencephalic and diencephalic areas initiates 22 kHz alarm vocalization. Therefore, the tegmental cholinergic neurons should be more active during emission of alarm calls. The aim of this study was to demonstrate increased activity of LDT cholinergic neurons during emission of 22 kHz calls induced by air puff stimuli. Immunohistochemical staining of the enzyme choline acetyltransferase identified cell bodies of cholinergic neurons, and c-Fos immunolabeling identified active cells. Double labeled cells were regarded as active cholinergic cells. There were significantly more (p<O.05) c-Fos labeled cells in the LDT of vocalizing animals than in control (non-vocalizing air puffed and naIve non-airpuffed) animals. Although the numbers were low, there were also significantly more (p<O.05) doublelabeled neurons in the LDT of vocalizing animals than in the non-vocalizing controls. Such a difference between vocalizing and control animals was not found in the neighbouring PPT nucleus. Results suggest that there are cholinergic and non-cholinergic cells, which are selectively active in the LDT during emission of 22 kHz alarm calls.
Ghrelin, a gut and brain peptide, is a potent stimulant for growth hormone (GH) secretion and feeding. Recent studies further show a critical role of ghrelin in the regulation of sleep-wakefulness. Laterodorsal tegmental nucleus (LDT), that regulates waking and rapid eye movement (REM) sleep, expresses GH secretagogue receptors (GHS-Rs). Thus, the present study was carried out to examine electrophysiological effects of ghrelin on LDT neurons using rat brainstem slices, and to determine the ionic mechanism involved. Whole cell recording revealed that ghrelin depolarizes LDT neurons dose-dependently in normal artificial cerebrospinal fluid (ACSF). The depolarization persisted in tetrodotoxin-containing ACSF (TTX ACSF), and is partially blocked by the application of [D-Lys3]-GHRP-6, a selective antagonist for GHS-Rs. Membrane resistance during the ghrelin-induced depolarization increased by about 18% than that before the depolarization. In addition, the ghrelin-induced depolarization was drastically reduced in high-K+ TTX ACSF with a K+ concentration of 13.25 mM. Reversal potentials obtained from I-V curves before and during the depolarization were about -83 mV, close to the equilibrium potential of the K+ channel. Most of the LDT neurons recorded were characterized by an A-current or both the A-current and a low threshold Ca2+ spike, and they were predominantly cholinergic. These results indicate that ghrelin depolarizes LDT neurons postsynaptically and dose-dependently via GHS-Rs, and that the ionic mechanisms underlying the ghrelin-induced depolarization include a decrease of K+ conductance. The results also suggest that LDT neurons are implicated in the cellular processes through which ghrelin participates in the regulation of sleep-wakefulness.
gamma-Aminobutyric acid (GABA)ergic neurons are widely distributed in brainstem structures involved in the regulation of the sleep-wake cycle, locomotion, and attention. These brainstem structures include the pedunculopontine nucleus (PPN), which is traditionally characterized by its population of cholinergic neurons that have local and wide-ranging connections. The functional heterogeneity of the PPN is partially explained by the topographic distribution of cholinergic neurons, but such heterogeneity might also arise from the organization of other neuronal populations within the PPN. To understand whether a topographical organization is also maintained by GABAergic neurons, we labeled these neurons by in situ hybridization for glutamic acid decarboxylase mRNA combined with immunohistochemistry for choline acetyltransferase to reveal cholinergic neurons. We analyzed their distribution within the PPN by using a method to quantify regional differences based on stereological cell counts. We show that GABAergic neurons of the rat PPN have a rostrocaudal gradient that is opposite to that of cholinergic neurons. Indeed, GABAergic neurons are predominantly concentrated in the rostral PPN; in addition, they form, along with cholinergic neurons, a small, high-density cluster in the most caudal portion of the nucleus. Thus, we provide evidence of heterogeneity in the distribution of different neuronal populations in the PPN and show that GABAergic and cholinergic neurons define neurochemically distinct areas. Our data suggest that the PPN is neurochemically segregated, and such differences define functional territories.
Orexin-A (ORX-A) and orexin-B (ORX-B) play critical roles in the regulation of sleep-wakefulness and feeding. ORX neurons project to the pedunculopontine tegmental nucleus (PPT), which regulates waking and rapid eye movement (REM) sleep. Thus, we examined electrophysiological effects of ORXs on rat PPT neurons with a soma size of more than 30 microm. Whole cell patch clamp recording in vitro revealed that ORX-A and ORX-B depolarized PPT neurons dose-dependently in normal and/or tetrodotoxin containing artificial cerebrospinal fluids (ACSFs), and the EC(50) values for ORX-A and ORX-B were 66 nM and 536 nM, respectively. SB-334867, a selective inhibitor for ORX 1 (OX(1)) receptors, significantly suppressed the ORX-A-induced depolarization. The ORX-A-induced depolarization was reduced in high K(+) ACSF with extracellular K(+) concentration of 13.25 mM or N-methyl-d-glucamine (NMDG(+))-containing ACSF in which NaCl was replaced with NMDG-Cl, and abolished in high K(+)-NMDG(+) ACSF or in a combination of NMDG(+) ACSF and recordings with Cs(+)-containing pipettes. An inhibitor of Na(+)/Ca(2+) exchanger and chelating intracellular Ca(2+) had no effect on the depolarization. Most of PPT neurons studied were characterized by an A-current or both A-current and a low threshold Ca(2+) spike, and predominantly cholinergic. These results suggest that ORXs directly depolarize PPT neurons via OX(1) receptors and via a dual ionic mechanism including a decrease of K(+) conductances and an increase of non-selective cationic conductances, and support the notion that ORX neurons affect the activity of PPT neurons directly and/or indirectly to control sleep-wakefulness, especially REM sleep.
Several studies have shown that the neuronal activity of the pedunculopontine nucleus is increased in Parkinson's disease. In the present study, the changes were examined in the firing rate and firing pattern of presumed cholinergic and non-cholinergic neurons in the pedunculopontine nucleus of 6-hydroxydopamine-lesioned rats by using extracellular recording. In the lesioned rats, the mean firing rate of both presumed cholinergic and non-cholinergic neurons in the pedunculopontine nucleus increased significantly compared to normal rats. With regard to firing pattern, the majority of presumed cholinergic and non-cholinergic neurons fired regularly in normal rats. After substantia nigra pars compacta-lesion, the percentage of presumed non-cholinergic neurons exhibiting irregular pattern increased significantly compared to normal rats, while having no significant change in the firing pattern of presumed cholinergic neurons. Collectively, these results indicate that the presumed cholinergic and non-cholinergic neurons in the pedunculopontine nucleus are overactive in 6-hydroxydopamine-lesioned rats, particularly, presumed non-cholinergic neuron firing is more irregular, which suggests that the firing activity of presumed cholinergic and non-cholinergic neurons is affected by the different afferents from the basal ganglia and related structures.
The pedunculopontine tegmental nucleus (PPTg) contains a population of cholinergic neurons (the Ch5 group) and non-cholinergic neurons. There appears to be functional interdigitation between these two groups, which both have extensive projections. The principal ascending connections are with thalamic nuclei and structures associated with the striatum, including the substantial nigra pars compacta. The descending connections are with a variety of nuclei in the pons, medulla and spinal cord, concerned with autonomic and motor functions. In the past, emphasis has been laid on the role of the PPTg in locomotion and behavioural state control. In this review, we emphasise the role of the PPTg in processing outputs from the striatum. The non-cholinergic neurons receive outflow from both dorsal and vental striatum, and lesions of the PPTg disrupt behaviour associated with each of these. Our review indicates that the PPTg is less concerned with the induction of locomotion and more concerned with relating reinforcement (information about which comes from the ventral striatum) with motor output from the dorsal striatum. The conclusions we draw are: (1) the PPTg is an outflow system for the striatum, but also forms a 'subsidiary circuit', returning information to striatal circuitry; in this, the PPTg has an anatomical organisation that resembles that of the substantia nigra. (2) As well as a role in the mediation of REM sleep, cholinergic PPTg neurons have an important role in the waking state, providing feedback into the thalamus and striatum. (3) The precise function of the computations performed on striatal outflow by the PPTg is uncertain. We discuss whether this function is complementary (parallel to other routes of striatal outflow), integrative (modifying other forms of striatal outflow) or both.
Halothane anesthesia causes spindles in the electroencephalogram (EEG), but the cellular and molecular mechanisms generating these spindles remain incompletely understood. The current study tested the hypothesis that halothane-induced EEG spindles are regulated, in part, by pontine cholinergic mechanisms.
Adult male cats were implanted with EEG electrodes and trained to sleep in the laboratory. Approximately 1 month after surgery, animals were anesthetized with halothane and a microdialysis probe was stereotaxically placed in the medial pontine reticular formation (mPRF). Simultaneous measurements were made of mPRF acetylcholine release and number of cortical EEG spindles during halothane anesthesia and subsequent wakefulness. In additional experiments, carbachol (88 mM) ws microinjected in the the mPRF before halothane anesthesia to determine whether enhanced cholinergic neurotransmission in the MPRF would block the ability of halothane to induce cortical EEG spindles.
During wakefulness, mPRF acetylcholine release averaged 0.43 pmol/10 min of dialysis. Halothane at 1 minimum alveolar concentration decreased acetylcholine release (0.25 pmol/10 min) while significantly increasing the number of cortical EEG spindles. Cortical EEG spindles caused by 1 minimum alveolar concentration halothane were not significantly different in waveform, amplitude, or number from the EEG spindles of nonrapid eye movement sleep. Microinjection of carbachol into the mPRF before halothane administration caused a significant reduction in number of halothane-induced EEG spindles.
Laterodorsal and pedunculopontine tegmental neurons, which provide cholinergic input to the mPRF, play a causal role in generating the EEG spindles of halothane anesthesia.
Tegmental cholinergic neurons vary their discharge patterns across the sleep-wake cycle, and glutamate is suggested to play an important role in determining these firing patterns. Cholinergic and noncholinergic neurons in the mesopontine tegmentum have different susceptibilities to various excitotoxins, presumably because of heterogeneity in the expression of glutamate receptor subtypes in this area. By using a double-labeling procedure that combines nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) histochemistry and avidin-biotin-peroxidase immunocytochemistry with diaminobenzidine as the chromogen, we compared the colocalization of AMPA receptor subunits GluR1, GluR2/3, and GluR4, kainate receptor subunits GluR5/6/7, and an NMDA receptor subunit NMDAR1 on NADPH-diaphorase-positive (cholinergic) neurons in the mesopontine tegmentum. Throughout the brainstem, neurons immunoreactive for GluR2/3 and NMDAR1 were most numerous, whereas neurons labeled for GluR1, GluR4, and GluR5/6/7 were less common. Specifically within the mesopontine tegmentum, the proportion of double-labeled neurons in the diaphorase-containing cell population was highest with GluR1 (43%) and lowest with GluR5/6/7 (12%). Regardless of the receptor subunit type, the greatest numbers of double-labeled neurons were observed in the pedunculopontine tegmental nucleus pars compacta and the fewest in the dorsal aspect of the laterodorsal tegmental nucleus. In addition, there were regional differences in the relative expression of receptor subunits and diaphorase-positive neurons across the subdivisions of the tegmental cholinergic column. Because each ionotropic subunit confers distinctive properties to a receptor channel, the present results suggest that mesopontine cholinergic neurons have nonuniform responses to glutamate and are also discriminable from basal forebrain cholinergic neurons in terms of glutamate receptor configuration.
Acetylcholine (ACh) in the medial pontine reticular formation (mPRF) originates from the laterodorsal and pedunculopontine tegmental (LDT/PPT) nuclei and contributes to generating rapid eye movement (REM) sleep. The mechanisms controlling mPRF ACh levels are incompletely understood. This study tested the hypothesis that mPRF ACh release is regulated, in part, by muscarinic autoreceptors. The mPRF of intact, halothane-anesthetized cats was dialyzed with Ringer's solution (control) or Ringer's containing the muscarinic antagonist scopolamine, Scopolamine caused a dose-dependent increase in mPRF ACh release and a concomitant decrease in the number of halothane-induced cortical EEG spindles. These data suggest that presynaptic muscarinic receptors, presumed to reside on cholinergic LDT/PPT terminals in the mPRF, play a role in regulating mPRF ACh release, REM sleep and EEG spindles.
The first objective of the present study was to verify whether projections from regions of the internal pallidum (GPi) that receive inputs from different functional areas of the striatum remain segregated at the level of the pedunculopontine nucleus (PPN) in squirrel monkeys. Second, we analyzed the ultrastructural features and synaptic organization of pallidal terminals in contact with PPN neurons. This was achieved by performing iontophoretic injections of biotinylated dextran amine (BDA) in different regions of the GPi.
Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirmation of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia. Astrocytic processes were stained in white matter, in the granular layet surrounding synaptic glomerular complexes, and in the molecular layer in the form of radially oriented fibers and of sheaths surrounding Purkinje cell dendrites. Astrocytic endfeet impinging on meninges and perivascular membranes were also antigen positive. In astrocytic perikarya and processes, the immunohistochemical reaction product appears both as a diffuse cytoplasmic label and as elongated strands, which by their distribution and frequency could be considered glial filaments.
Converging lines of evidence suggest that the hypothalamic suprachiasmatic nucleus (SCN) is the site of the endogenous biological clock controlling mammalian circadian rhythms. To study the calcium responses of the cellular components that make up the clock, computer-controlled digital video and confocal scanning laser microscopy were used with the Ca2+ indicator dye fluo-3 to examine dispersed SCN cells and SCN explants with repeated sampling over time. Ca2+ plays an important second messenger role in a wide variety of cellular mechanisms from gene regulation to electrical activity and neurotransmitter release, and may play a role in clock function and entrainment. SCN neurons and astrocytes showed an intracellular Ca2+ increase in response to glutamate and 5-HT, two major neurotransmitters in afferents to the SCN. Astrocytes showed a marked heterogeneity in their response to the serial perfusion of different transmitters; some responded to both 5-HT and glutamate, some to neither, and others to only one or the other. Under constant conditions, most neurons showed irregular temporal patterns of Ca2+ transients. Expression of regular neuronal oscillations could be blocked by the inhibitory transmitter GABA. Astrocytes, on the other hand, showed very regular rhythms of cytoplasmic Ca2+ concentrations with periods ranging from 7 to 20 sec. This periodic oscillation could be initiated by in vitro application of glutamate, the putative neurotransmitter conveying visual input to the SCN critical for clock entrainment. Long-distance communication between glial cells, seen as waves of fluorescence moving from cell to cell, probably through gap junctions, was induced by glutamate, 5-HT, and ATP. These waves increased the period length of cellular Ca2+ rises to 45-70 sec. Spontaneously oscillating cells were common in culture medium, serum, or rat cerebrospinal fluid, but rare in HEPES buffer. One source for cytoplasmic Ca2+ increases was an influx of extracellular Ca2+, as seen under depolarizing conditions in about 75% of the astroglia studied. All neurotransmitter-induced Ca2+ fluxes were not dependent on voltage changes, as Ca2+ oscillations could be initiated under both normal and depolarizing conditions. Since neurotransmitters could induce a Ca2+ rise in the absence of extracellular Ca2+, the mechanisms of ultradian oscillations appear to depend on cycles of intracellular Ca2+ fluxes from Ca(2+)-sequestering organelles into the cytoplasm, followed by a subsequent Ca2+ reduction. In the adult SCN, fewer astrocytes are found than neurons, yet astrocytes frequently surround glutamate-immunoreactive axons in synaptic contact with SCN dendrites, isolating neurons from each other while maintaining contact with other astrocytes by gap junctions.(ABSTRACT TRUNCATED AT 400 WORDS)
Serotonergic suppression of cholinergic neuronal activity implicated in the regulation of rapid eye movement sleep and its associated phenomenon, pontogeniculooccipital waves, has long been postulated, but no direct proof has been available. In this study, intracellular and whole-cell patch-clamp recording techniques were combined with enzyme histochemistry to examine the intrinsic electrophysiological properties and response to serotonin (5-HT) of identified cholinergic rat laterodorsal tegmental nucleus neurons in vitro. Sixty-five percent of the recorded neurons demonstrated a prominent low-threshold burst, and of these, 83% were cholinergic. In current-clamp recordings 64% of the bursting cholinergic neurons tested responded to the application of 5-HT with a membrane hyperpolarization and decrease in input resistance. This effect was mimicked by application of the selective 5-HT type 1 receptor agonist carboxamidotryptamine maleate. Whole-cell patch-clamp recordings revealed that the hyperpolarizing response was mediated by an inwardly rectifying K+ current. Application of 5-HT decreased excitability and markedly modulated the discharge pattern of cholinergic bursting neurons: during a 5-HT-induced hyperpolarization these neurons exhibited no rebound burst after hyperpolarizing current input and a burst in response to depolarizing current input. In the absence of 5-HT, the relatively depolarized cholinergic bursting neurons responded to an identical hyperpolarizing current input with a burst and did not produce a burst after depolarizing current input. These data provide a cellular and molecular basis for the hypothesis that 5-HT modulates rapid eye movement sleep phenomenology by altering the firing pattern of bursting cholinergic neurons.
As originally named for the ostensibly contradictory appearance of rapid eye movements and low voltage fast cortical activity during behavioral sleep, paradoxical sleep or rapid eye movement sleep, represents a distinct third state, in addition to waking and slow wave sleep, in mammals and birds. It is an internally generated state of intense tonic and phasic central activation that is contemporaneous with the inhibition of sensory input and motor output. In early studies, it was established that the state of paradoxical sleep was generated within the brainstem, and particularly within the pons. Pharmacological studies indicated an important role for acetylcholine as a neurotransmitter in the generation of this state. Local injections of cholinergic agonists into the pontine tegmentum triggered a state of paradoxical sleep marked by phasic ponto-geniculo-occipital spikes in association with cortical activation and neck muscle atonia. Following the immunohistochemical identification of choline acetyl transferase-containing neurons and their localization to the dorsolateral ponto-mesencephalic tegmentum, neurotoxic lesions of this major cholinergic cell group could be performed to assess its importance in paradoxical sleep. Destruction of the majority of the cholinergic cells, which are concentrated within the laterodorsal tegmental and pedunculopontine tegmental nuclei but extend also into the locus coeruleus and parabrachial nuclei in the cat, resulted in a loss or diminishment of the state of paradoxical sleep, ponto-geniculo-occipital spiking and neck muscle atonia. These deficits were correlated with the loss of choline acetyltransferase-immunoreactive neurons in the region, so as to corroborate results of pharmacological studies and single unit recording studies indicating an active role of these cholinergic cells in the generation of paradoxical sleep and its components. These cells provide a cholinergic innervation to the entire brainstem reticular formation that may be critical in the generation of the state which involves recruitment of massive populations of reticular neurons. Major ascending projections into the thalamus, including the lateral geniculate, may provide the means by which phasic (including ponto-geniculo-occipital spikes) and tonic activation is communicated in part to the cerebral cortex. Descending projections through the caudal dorsolateral pontine tegmentum and into the medial medullary reticular formation may be involved in the initiation of sensorimotor inhibition. Although it appears that the pontomesencephalic cholinergic neurons play an important, active role in the generation of paradoxical sleep, this role may be conditional upon the simultaneous inactivity of noradrenaline and serotonin neurons, evidence for which derives from both pharmacological and recording studies.(ABSTRACT TRUNCATED AT 400 WORDS)
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 afferent input to the basal forebrain cholinergic neurons from the pontomesencephalic tegmentum was examined by retrograde transport of wheatgerm agglutinin-horseradish peroxidase in combination with immunohistochemistry. Multiple tyrosine hydroxylase-, dopamine-beta-hydroxylase-, serotonin- and choline acetyltransferase-immunoreactive fibres were observed in the vicinity of the choline acetyltransferase-immunoreactive cell bodies within the globus pallidus, substantia innominata and magnocellular preoptic nucleus. Micro-injections of horseradish peroxidase-conjugated wheatgerm agglutinin into this area of cholinergic perikarya led to retrograde labelling of a large population of neurons within the pontomesencephalic tegmentum, which included cells in the ventral tegmental area, substantia nigra, retrorubral field, raphe nuclei, reticular formation, pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus, parabrachial nuclei and locus coeruleus nucleus. Of the total population of retrogradely labelled neurons, a significant (approximately 25%) proportion were tyrosine hydroxylase-immunoreactive and found in the ventral tegmental area (A10), the substantia nigra (A9), the retrorubral field (A8), the raphe nuclei (dorsalis, linearis and interfascicularis) and the locus coeruleus nucleus (A6), Another important contingent (approximately 10%) was represented by serotonin neurons of the dorsal raphe nucleus (B7), the central superior nucleus (B8) and ventral tegmentum (B9). A small proportion (less than 1%) was represented by cholinergic neurons of the pedunculopontine (Ch5) and laterodorsal (Ch6) tegmental nuclei. These results demonstrate that pontomesencephalic monoamine neurons project in large numbers up to the basal forebrain cholinergic neurons and may represent a major component of the ventral tegmental pathway that forms the extra-thalamic relay from the brainstem through the basal forebrain to the cerebral cortex.
Kainic acid was injected bilaterally (4.8 micrograms in 1.2 microliters each side) into the dorsolateral pontomesencephalic tegmentum of cats in order to destroy the cholinergic neurons located in that region and thus to study the effects of their destruction upon sleep-waking states. The kainic acid produced a large area of nerve cell loss and/or gliosis centered in the dorsolateral tegmentum-cholinergic cell area, that includes the pedunculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei rostrally (A1-P2), and the parabrachial (PB) and locus coeruleus (LC) nuclei caudally (P3-P5). The mean loss of choline acetyltransferase (ChAT)-immunoreactive neurons within this area was 60% with a range from 25% to 85% across 11 cats. The mean loss of tyrosine hydroxylase (TH)-immunoreactive neurons, differentially distributed through the same region, was 35% with a range of 0-50%. Whereas the kainic acid lesions appeared to have only slight effects upon wakefulness and slow-wave sleep, they had marked effects upon paradoxical sleep (PS), which varied in degree across animals. In cats with the most extensive destruction of cholinergic neurons, PS was eliminated in the first few weeks following the lesion and then reappeared as isolated episodes characterized by sparse, low amplitude PGO spikes in association with few eye movements and an activated cortex, though in absence of neck muscle atonia. Although these PS-like episodes varied in amount, they were significantly less than baseline PS in percent and in duration for the group of 11 animals over one month recording. The PGO spike rate was significantly reduced; the EMG amplitude was significantly increased, marking a loss of neck muscle atonia. The percent of PS-like epochs, the rate of PGO spiking and the EMG amplitude on postlesion day 28 were found to be significantly correlated with the volume of the lesion within the dorsolateral pontine tegmentum-cholinergic cell area. The percent PS-like episodes and PGO spike rate were significantly correlated with the number of remaining ChAT-immunoreactive neurons, but not with the number of remaining TH-immunoreactive neurons within this region. These results suggest that cholinergic pontomesencephalic neurons may be critically involved in the generation of paradoxical sleep and its phasic events.
Sleep is characterized by synchronized events in billions of synaptically coupled neurons in thalamocortical systems. The activation of a series of neuromodulatory transmitter systems during awakening blocks low-frequency oscillations, induces fast rhythms, and allows the brain to recover full responsiveness. Analysis of cortical and thalamic networks at many levels, from molecules to single neurons to large neuronal assemblies, with a variety of techniques, ranging from intracellular recordings in vivo and in vitro to computer simulations, is beginning to yield insights into the mechanisms of the generation, modulation, and function of brain oscillations
Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirmation of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia. Astrocytic processes were stained in white matter, in the granular layet surrounding synaptic glomerular complexes, and in the molecular layer in the form of radially oriented fibers and of sheaths surrounding Purkinje cell dendrites. Astrocytic endfeet impinging on meninges and perivascular membranes were also antigen positive. In astrocytic perikarya and processes, the immunohistochemical reaction product appears both as a diffuse cytoplasmic label and as elongated strands, which by their distribution and frequency could be considered glial filaments.
A monograph communicating the current realities and future possibilities of unifying basic studies on anatomy and cellular physiology with investigations of the behavioral and physiological events of waking and sleep. Steriade established the Laboratory of Neurophysiology at Laval U., Quebec; McCarl
The nucleus basalis of Meynert in the squirrel monkey exhibits numerous labeled neurons following the retrograde transport of horseradish perox-idase from occipital cortical injection sites. The typically large, often clustered, labeled cells are seen most frequently in association with the fibrous bordering structures of the substantia innominata and in the internal and external laminae of the globus pallidus. Ultrastructurally the copious cytoplasm of nucleus basalis neurons abounds with organelles. Large, vacuo-lated lipofuscin granules proliferate as a function of age and are not evident in younger monkeys. Approximately 4% of the somal surface is occupied by symmetrical synapses with either flat or pleomorphic vesicles. The remainder is covered mostly by neuroglial processes. Somatic spines bearing synapses are occasionally observed. In the neuropil surrounding nucleus ba-salis somata, the synapses onto dendrites and spines are mostly asymmetrical with large, round vesicles. Labeled nucleus basalis cells in the substantia innominata immediately lateral to the optic tract are larger and rounder than cells in the internal and external pallidal laminae. However, no remarkable ultrastructural differences were observed between nucleus basalis so-mata in the substantia innominata and external pallidal lamina, or between horseradish peroxidase-labeled and unlabeled large cells.
The laterodorsal tegmental nucleus (LDT) is the largest aggregation in the brainstem of cholinergic neurons whose axons reach the thalamus as part of a diffuse projection to the forebrain. We measured the regional blood flow in the thalamus by means of laser Doppler flowmetry, and examined whether the blood flow was regulated by the ascending cholinergic nerve fibers originating in the LDT. Experiments were performed on urethane-anesthetized rats whose upper cervical spinal cord was transected to avoid response of systemic blood pressure following LDT stimulation. The ascending cholinergic nerve fibers were excited by electrical or chemical stimulation applied to the LDT. The regional thalamic blood flow increased in response to repetitive electrical stimulation and chemical stimulation with L-glutamate to the LDT. The response, starting several seconds after the onset of electrical stimulation and lasting as long as 1 min, was reduced by i.v. scopolamine, a cholinergic muscarinic receptor antagonist. The results indicate that regional blood flow in the thalamus is increased by excitation of the ascending cholinergic nerve fibers originating in the LDT mainly through the cholinergic muscarinic receptors.
Compelling evidence indicates that cholinergic basal forebrain neurons are strongly activated during waking, and concurrently thalamic spindle activity is suppressed and thalamocortical sensory transmission is facilitated. Both thalamus and basal forebrain are known to receive projections from brainstem cholinergic and aminergic neuronal pools that are involved in wake/sleep regulation. The present study addressed the question of whether single cholinergic and aminergic neurons contributed to both of these ascending projections, by using two fluorescent retrograde tracers combined with immunoflurrescence. Cholinergic neurons projecting to both the basal forebrain and thalamus were found in the pedunculopontine and laterodorsal tegmental nuclei, representing an average of 8.0% of the total cholinergic cell population in these nuclei. Serotonergic neurons with dual projections were observed in the dorsal, median and caudal linear raphe nuclei, accounting for a mean of 4.7% of total serotonergic nerrons in these nuclei. Relatively few noradrenergic neurons (2.0%) in the locus ceruleus projected to both target structures, and a very small subpopulation of histaminergic neurons (1.5%) in the tuberomammillary hypothalamic nucleus had dual projections. Of all brainstem neurons with dual projections, cholinergic and serotonergic neurons accounted for an overwhelming majority, with noradrenergic followed by histaminergic neurons representing the remaining minority. These data suggest that through dual projections, cholinergic and aminnrgic brainstem neurons can concurrently modulate the activity of neurons in the thalamus and basal forebrain during cortical arousal.
A major group of cholinergic neurons is present in the midbrain and pontine tegmentum. These cells could be selectively stained using either monoclonal antibodies to choline acetyltransferase, the pharmacohistochemical acetylcholinesterase procedure, or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Using these three techniques, the precise distribution of this cell group was determined. By combining these techniques with immunohistochemical staining for various neuropeptides, examples of peptide-cholinergic coexistence could be demonstrated in this cell group. Approximately 30% of these cholinergic neurons displayed substance P immunoreactivity. Most of these cells also showed corticotropin-releasing factor immunoreactivity and bombesin/gastrin-releasing peptide immunoreactivity. These results therefore provide evidence for the coexistence of various neuropeptides together with NADPH-diaphorase activity in the ascending cholinergic reticular system.
With the indirect immunofluorescence technique, the localization (including the postnatal ontogeny) of substance P in the cerebellum, and the ways of entry of substance P-containing fibers into the cerebellum were explored. In the newborn rat cerebellum, dense fiber bands of axons with substance P-like immunoreactivity which can be traced to the lower brain stem are found. These fibers are also traceable to the developing granular cell layer. Two weeks after birth, however, substance P-containing structures seen in the cerebellum begin to decrease progressively and in the cerebellum of the adult rats, only a small amount of substance P-containing structures is observed. The present study established that substance P-containing fibers are mostly derived from extracerebellar substance P-containing cells and demonstrated the presence of three sites of entry of these substance P-containing fibers to the cerebellum, via (1) the inferior cerebellar peduncle, (2) the fasciculus uncinatus and (3) the middle cerebellar peduncle, respectively. Following deafferentation of the cerebellum, substance P-accumulating fibers are observed only ventral to the lesion (i.e. on the brain stem side), while in the cerebellum a remarkable decrease of substance P-containing fibers is seen and no substance P-accumulating fibers are found dorsal to the lesion (cerebellar side).
University Microfilms order no. UMI00359154. Thesis (Ph. D.)--University of British Columbia, 1991. Includes bibliographical references.
During the sleep cycle in cats, neurons localized to the posterolateral pole of the nucleus locus coeruleus and the nucleus
subcoeruleus undergo discharge rate changes that are the opposite of those of the pontine reticular giant cells. The inverse
rate ratios and activity curves of these two interconnected populations are compatible with reciprocal interaction as a physiological
basis of sleep cycle oscillation.
Increasingly strong evidence suggests that cholinergic neurons in the mesopontine tegmentum play important roles in the control of wakefulness and sleep. To understand better how the activity of these neurons is regulated, the potential afferent connections of the laterodorsal (LDT) and pedunculopontine tegmental nuclei (PPT) were investigated in the rat. This was accomplished by using retrograde and anterograde axonal transport methods and NADPH-diaphorase histochemistry. Immunohistochemistry was also used to identify the transmitter content of some of the retrogradely identified afferents.
The topographic arrangement of globus pallidus neurons sending axons to the subthalamic nucleus, auditory cortex and pedunculopontine tegmental nucleus was studied in the rat using retrograde fluorescent tracers. Neurons projecting to the subthalamic nucleus were localized in the rostral part of the globus pallidus, while neurons projecting to the auditory cortex and to the pedunculopontine tegmental nucleus were located in the caudal part. The two populations of pallidocortical and pallidotegmental neurons were also distributed in a separate manner within the caudal globus pallidus. The former neurons were large and located more ventromedially, whereas the latter were medium-sized and located more dorsolaterally. Using a retrograde fluorescent tracing technique combined with choline acetyltransferase immunofluorescence histochemistry, it was found that a vast majority of pallidocortical neurons expressed choline acetyltransferase immunoreactivity, and that pallidotegmental neurons rarely exhibited choline acetyltransferase immunoreactivity. A method of retrograde tracing with wheatgerm agglutinin conjugated with horseradish peroxidase associated to immunohistochemistry for glutamate decarboxylase confirmed the GABAergic nature of the pallidotegmental pathway. The present study revealed the independent nature of the globus pallidus neurons projecting to the subthalamic nucleus, auditory cortex and pedunculopontine tegmental nucleus. Within this cellular arrangement, the presence of functionally distinct neuronal populations at the caudal pallidal level was also identified, with large cholinergic cells innervating the neocortex and medium-sized GABAergic cells "feeding" the mesencephalic tegmentum.
The afferent connections of the pedunculopontine tegmental nucleus (PPT) and the adjacent midbrain extrapyramidal area (MEA) were examined by retrograde tracing with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Major afferents to the PPT originate in the periaqueductal gray, central tegmental field, lateral hypothalamic area, dorsal raphe nucleus, superior colliculus, and pontine and medullary reticular fields. Other putative inputs originate in the paraventricular and preoptic hypothalamic nuclei, the zona incerta, nucleus of the solitary tract, central superior raphe nucleus, substantia innominata, posterior hypothalamic area, and thalamic parafascicular nucleus. The major afferent to the medially adjacent MEA originates in the lateral habenula, while other putative afferents include the perifornical and lateral hypothalamic area, periaqueductal gray, superior colliculus, pontine reticular formation, and dorsal raphe nucleus. MEA inputs from basal ganglia nuclei include moderate projections from the substantia nigra pars reticulata, entopeduncular nucleus, and a small projection from the globus pallidus, but not the subthalamic nucleus. Dense anterograde labeling was observed in the substantia nigra pars compacta, entopeduncular nucleus, subthalamic nucleus, globus pallidus, and caudate-putamen only following WGA-HRP injections involving the MEA.
The ultrastructure of choline acetyltransferase (ChAT)-immunoreactive neurons in the laterodorsal tegmental nucleus (TLD) of the rat was investigated by immunohistochemical techniques. The immunoreactive neurons were medium to large in size, with a few elongated dendrites, contained well-developed cytoplasm, and a nucleus with deep infoldings. They received many nonimmunoreactive, mostly asymmetric synaptic inputs on their soma and dendrites. ChAT-immunoreactive, usually myelinated, axons were occasionally seen in TLD. Only one immunoreactive axon terminal was observed within TLD, and it made synaptic contact with a nonimmunoreactive neuronal perikaryon. The synaptic interactions between ChAT-immunoreactive neurons and tyrosine hydroxylase (TH)-immunoreactive fibers in the TLD were investigated with a double immunohistochemical staining method. ChAT-immunoreactivity detected with a beta-galactosidase method was light blue-green in the light microscope and formed dot-like electron dense particles at the electron microscopic level. TH-immunoreactivity, visualized with a nickel-enhanced immunoperoxidase method, was dark blue-black in the light microscope and diffusely opaque in the electron microscope. Therefore, the difference between these two kinds of immunoreactivity could be quite easily distinguished at both light and electron microscopic levels. In the light microscope, TH-positive fibers were often closely apposed to ChAT-immunoreactive cell bodies and dendrites in TLD. In the electron microscope, the cell soma and proximal dendrites of ChAT-immunoreactive neurons received synaptic contacts from TH-immunoreactive axon terminals. These results provide a morphological basis for catecholaminergic regulation of the cholinergic reticular system.
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.
Transection, lesion and unit recording studies have localized rapid eye movement (REM) sleep mechanisms to the pons. Recent work has emphasized the role of pontine cholinergic cells, especially those of the pedunculopontine tegmentum (PPT). The present study differentiated REM sleep deficits associated with lesions of the PPT from other pontine regions implicated in REM sleep generation, including those with predominantly cholinergic vs non-cholinergic cells. Twelve hour polygraphic recordings were obtained in 18 cats before and 1-2 weeks after bilateral electrolytic or radio frequency lesions of either: (1) PPT, which contains the dorsolateral pontine cholinergic cell column; (2) laterodorsal tegmental nucleus (LDT), which contains the dorsomedial pontine cholinergic cell column; (3) locus ceruleus (LC), which contains mostly noradrenergic cells; or (4) subceruleus (LC alpha, peri-LC alpha and the lateral tegmental field), which also contains predominantly noncholinergic cells. There were three main findings: (i) Only lesions of PPT and subceruleus significantly affected REM sleep time. These lesions produced comparable reductions in REM sleep time but influenced REM sleep components quite differently: (ii) PPT lesions, estimated to damage 90 +/- 4% of cholinergic cells, reduced the number of REM sleep entrances and phasic events, including ponto-geniculooccipital (PGO) spikes and rapid eye movements (REMs), but did not prevent complete atonia during REM sleep: (iii) Subceruleus lesions eliminated atonia during REM sleep. Mobility appeared to arouse the cat prematurely from REM sleep and may explain the brief duration of REM sleep epochs seen exclusively in this group. Despite the reduced amount of REM sleep, the total number of PGO spikes and REM sleep entrances increased over baseline values. Collectively, the results distinguish pontine loci regulating phasic events vs atonia. PPT lesions reduced phasic events, whereas subceruleus lesions created REM sleep without atonia. Severe REM sleep deficits after large pontine lesions, including PPT and subceruleus, might be explained by simultaneous production of both REM sleep syndromes. However, extensive loss of ACh neurons in the PPT does not disrupt REM sleep atonia.
The connections of the laterodorsal tegmental nucleus (LDTg) have been investigated using anterograde and retrograde lectin tracers with immunocytochemical detection. Inputs to LDTg were found from frontal cortex, diagonal band, preoptic areas, lateral hypothalamus, lateral mamillary nucleus, lateral habenula; the interpeduncular nucleus, ventral tegmental area, substantia nigra and retrorubral fields; the medial terminal nucleus, interstitial nucleus, supraoculomotor central grey, medial pretectum, nucleus of the posterior commissure, paramedian pontine reticular formation, paraabducens and paratrochlear region; the parabrachial nuclei and nucleus of the tractus solitarius. Terminal labelling from PHA-L injections of LDTg was found in infralimbic, cingulate and hippocampal cortex, lateral septum, septofimbrial and triangular nuclei, horizontal limb of diagonal band and preoptic areas; in the anterior, mediodorsal, reuniens, centrolateral, parafascicular, paraventricular and laterodorsal thalamic nuclei, rostral reticular thalamic nucleus, and zona incerta; the lateral habenula and the lateral hypothalamus. A number of brainstem structures apparently associated with visual functions were also innervated, mainly the superior colliculus, medial pretectum, medial terminal nucleus, paramedian pontine reticular formation, inferior olive, supraoculomotor, paraabducens and supragenual regions, prepositus hypoglossi and nucleus of the posterior commissure. Also innervated were substantia nigra compacta, ventral tegmental area, interfascicular nucleus, interpeduncular nucleus, dorsal and medial raphe, pedunculopontine tegmental region, parabrachial nuclei, and nucleus of the tractus solitarius. These findings suggest the LDTg to be a highly differentiated part of the ascending "reticular activating" system, concerned not only with specific cortical and thalamic regions, especially those associated with the limbic system, but also with the basal ganglia, and visual (particularly oculomotor) mechanisms. Additional links with the habenula-interpeduncular system are discussed in this context.
Nitric oxide (NO) is a recently discovered and highly unorthodox messenger molecule. Current evidence indicates that, in the CNS, NO is produced enzymatically in postsynaptic structures in response to activation of excitatory amino acid receptors. It then diffuses out to act on neighbouring cellular elements, probably presynaptic nerve endings and astrocyte processes. In several peripheral nerves, and quite possibly in parts of the CNS as well, NO might be formed presynaptically and thus act as a neurotransmitter. In both cases, a major action of NO is to activate soluble guanylate cyclase and so raise cGMP levels in target cells.
Acetylcholine (ACh) has long been implicated in the regulation of arousal or wakefulness. However, the anatomical basis for this regulation had been missing because relatively little was known about the organization of central cholinergic pathways. During the last decade, however, specific immuno-histochemical markers became available, and by using these markers central cholinergic neurons have been mapped and their projections delineated (see Semba and Fibiger, 1989, for review). It is now well established that there are two major cholinergic projection systems in the CNS: cholinergic neurons in the basal forebrain project widely to the cerebral cortex, and those in the mesopontine tegmentum project heavily to the thalamus. Armed with these anatomical findings, researchers of behavioral state have begun to investigate the role of specific populations of central cholinergic neurons in the regulation of waking and sleep. One important conclusion which has emerged from such recent studies is that cholinergic neurons in the basal forebrain have a crucial role in cortical arousal. In the present paper, both anatomical and physiological evidence supporting this notion is discussed, and clues are explored as to how the activity of basal forebrain cholinergic neurons is regulated during different behavioral states.
Microinjection of cholinergic agonists and acetylcholinesterase inhibitors into the medial pontine reticular formation (mPRF) causes a state that is polygraphically similar to rapid-eye-movement (REM) sleep. Respiratory studies of intact unanesthetized cats during this cholinergically induced REM sleep-like state have shown that the same cholinoceptive pontine reticular regions that mediate REM sleep can also cause state-dependent respiratory depression. The present study investigated the hypothesis that acetylcholine (ACh) release in the mPRF is increased during the respiratory depression that accompanies the cholinergically induced REM sleep-like state. Cats were implanted for polygraphic recording of sleep and wakefulness and with guide tubes aimed for placing a microinjector in one mPRF and a microdialysis probe in the contralateral mPRF. ACh release was measured with high-performance liquid chromatography and electrochemical detection. Compared with waking levels, ACh was significantly increased and respiratory frequency was significantly decreased during the carbachol-induced REM sleep-like state. These results support the hypothesis that endogenous cholinergic neurotransmission in brain regions known to regulate REM sleep can also cause state-dependent changes in respiratory control.
Although sleep-wake cycles are repeated every day and night and almost one-third of our lifetime is spent sleeping, the molecular mechanisms of sleep-wake regulation have remained little understood. Recent experimental evidence indicates that prostaglandins (PG) D2 and E2 are probably two of the major endogenous sleep-regulating substances, one promoting sleep and the other wakefulness, in rats, dogs, rabbits, monkeys, and probably in humans as well. Preliminary evidence indicates that the sites of action of PGD2 and E2 are located in the sleep and wake centers in or near the preoptic area and posterior hypothalamus, respectively.
The effects of microinjections of a cholinergic agonist, carbachol (0.2 microgram/0.2 microliter), were examined on three different types of rostrally projecting tonic neurons that we have reported previously in the dorsal part of the pontomesencephalic tegmentum known to contain numerous cholinergic cell bodies: 1) tonic type I slow (Type I-S); 2) tonic type I rapid (Type I-R); and 3) tonic type II (Type II) (El Mansari et al. 1989). Microinjections of carbachol near unit recording sites in freely moving cats induced within a few minutes a complete suppression of the spontaneous activity and a marked reduction in orthodromic excitation of identified and non-identified type I-S neurons. These effect lasted for approximately 90-120 min and were reversed by local (0.4 microgram/0.2 microliter) or systemic (0.1-0.2 mg/kg, i.m.) administration of atropine sulfate. In contrast, the cholinergic agonist had no consistent effects on tonic type II nor on tonic type I-R neurons. In the light of these and other recent findings, we suggested the direct inhibition of central cholinergic neurons via muscarinic receptors, on the one hand, and the cholinergic nature of type I-S, but not type I-R nor type II neurons, on the other.
Carbachol, a long-acting cholinergic agonist, was microinjected (4 micrograms/250 nl per 90 s) into 90 sites within the anterodorsal pontine tegmentum of four cats and the time to onset and percentage of time spent in a desynchronized sleep-like state during 40 min postinjection were calculated. Compared with more posteroventral pontine sites, the shorter latencies and higher percentages observed confirmed earlier predictions of a sensitive cholinoceptive zone in the anterodorsal pons. In 27 trials a desynchronized sleep-like state was observed within 5 min; in 31 trials the latency was 5-10 min and in the remaining 32 trials, greater than 10 min. Plotting the desynchronized sleep-like state latency and the desynchronized sleep-like state percentage as a function of the three-dimensional coordinates revealed that injection sites with short latency (less than 5 min) and high percentage (greater than 80%) were concentrated between the coordinates of P 1.0 to 3.5 and V -3.5 to -5.5, at the lateral coordinate L 2.0. On the frontal plane, the short desynchronized sleep-like state latency and high desynchronized sleep-like state percentage sites begin in the pontine tegmental region just lateral to the ventral tegmental nucleus and extend 3 mm ventrocaudally. A regression plot of the data in sagittal plane 2.0 revealed a short latency axis, around which the short latency sites cluster, running in a slightly dorsoventral direction from about P 1.0 to V -4.0 to P 4.0 to V -5.5. This observation suggests that the sensitive zone might approximate a cylinder in shape, a hypothesis supported by the correlation of longer latencies and lower percentages at increasing radial distance from the axis. The non-linear relationship between cholinergic potency and distance from the short latency axis suggests that the desynchronized sleep-like state latency is a function of two factors; a variable diffusion-based delay of carbachol to distant neuronal populations involved in the desynchronized sleep-like state production, and a fixed recruitment-based delay following activation of neurons in the sensitive zone. Interpretation of these findings in light of earlier studies involving microstimulation of the pontine tegmentum argue in favor of a distributed network of discrete neuronal populations as the source of desynchronized sleep generation.
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.
Membrane properties and postsynaptic responses to stimulation of the substantia nigra reticulata (SNr) of the neurons in rat pedunculopontine nucleus (PPN) were studied in an in vitro parasagittal slice preparation using intracellular recording techniques. Based on electrical membrane properties, PPN neurons were classified into 3 types (types I, II and II). The unique feature of the type I neuron was the low threshold calcium spike while the type II neuron had various inward and outward rectifications. The type III neuron showed no such features as those observed in type I or II neurons. Some recorded neurons were intracellularly labeled with biocytin to study their morphology, and their transmitter phenotype was investigated by immunocytochemistry for choline acetyltransferase (ChAT). The type I and III neurons were found to be non-cholinergic, but 50% of the labeled type II neurons were immunopositive for ChAT. Morphological features of type II neurons were also different from type I or III neurons. The soma of the type II neuron was almost always more than twice as large as that of type I and III neurons. Inhibitory postsynaptic potentials (IPSPs) were induced in all 3 types of PPN neurons following stimulation of SNr. SNr-induced IPSPs were usually followed by a slow depolarizing potential from which rebound spikes were triggered. These rebound excitations were found only in type I and II neurons. These data indicate that heterogeneous groups of neurons exist in the PPN in terms of morphology, transmitter phenotypes and electrical membrane properties.
Studies of the pedunculopontine (PPT) and laterodorsal tegmental (LDT) nuclei in the mesopontine tegmentum have emphasized the organization and projections of the cholinergic neurons. We report here that exhibiting glutamate immunoreactivity are present in both the LDT and PPT. These glutamatergic neurons are interspersed among the cholinergic neurons within both nuclei with no apparent segregation. These data raise the possibility that excitatory amino acids contribute to the functions of the LDT and PPT.
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.
In this study we determined that cholinergic neurons from the lateral dorsal tegmental (LDT) and peribrachial pontine region (PPG) innervate the medial pontine reticular formation (medial PRF), a region involved in the generation of REM sleep. Wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the medial PRF and the brainstem tissue was processed using a combined retrograde transport/immunocytochemical procedure. Results showed that 10-15% of choline acetyltransferase (ChAT) immunoreactive neurons in the LDT and PPG project to the medial PRF. It is hypothesized that these neurons play an important role in the generation of the REM sleep state.
The cholinergic agonist carbachol was conjugated to latex microspheres that were fluorescently labeled with rhodamine and
used as neuroanatomical probes that show little diffusion from their injection site and retrogradely label neurons projecting
to the injection site. Microinjection of this pharmacologically active probe into the gigantocellular field of the cat pontine
brain stem caused the awake cats to fall into rapid movement (REM) sleep indistinguishable from that produced by free carbachol.
Three-dimensional computer reconstruction of the retrogradely labeled neurons revealed a widely distributed neuronal network
in the pontine tegmentum. These pharmacologically active microspheres permit a new precision in the characterization and mapping
of neurons associated with the control of behavioral state and of other cholinergic networks.
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.
This chapter provides an update and overview of the anatomy of central cholinergic systems, and discusses some areas about which controversy and uncertainty still exist. Cholinergic neurons in the basal forebrain are distributed across several classically defined nuclei, including the medial septa1 nucleus, the nucleus of the diagonal band of Broca (vertical and horizontal limbs), the magnocellular preoptic area, the substantia innominata, and the globus pallidus. Many laboratories have studied the anatomy of these magnocellular neurons and there appears to be widespread agreement about their distribution in the basal forebrain. On the basis of connectivity patterns, the cholinergic neurons in the basal forebrain can be subdivided into four groups. Chl and Ch2 represent cholinergic neurons within the medial septal nucleus and the vertical limb of the diagonal band and provide the major source of cholinergic afferents to the hippocampus. Ch3 is contained primarily within the lateral part of the horizontal limb of the diagonal band, including the magnocellular preoptic area, and is the major source of cholinergic activity in the olfactory bulb. Ch4 consists of cholinergic neurons that are defined by the fact that they project to the neocortical mantle.
The topographical distribution, histochemical characteristics, and anatomical relationships of the cellular elements containing choline acetyltransferase (ChAT) immunoreactivity, demonstrated with specific monoclonal antibodies to ChAT following the unlabelled antibody peroxidase-antiperoxidase (PAP) procedure at the optical and electron microscopic levels, were investigated in the rat substantia nigra (SN).
Scarce, large (20–30 μm in maximum soma extent) cholinergic cell bodies and processes were found within the boundaries of the SN, in the borders of the pars compacta and pars reticulata, principally at caudal levels. Occasionally, cholinergic neurons were also found at intermediate levels of the SN, in the borders of the pars reticulata and pars lateralis. Cytologically, these large cells resembled ChAT-positive neurons localized in other areas of the central nervous system (CNS) of the rat—for example, the pontomesencephalotegmental (PMT) cholinergic complex (Ch5-Ch6) and the nucleus basalis of Meynert (nbM) (Ch4).
Histochemically, ChAT-positive cells in the SN were characterized by their ability to utilize the reduced cofactor nicotinamide adenine dinucleotide phosphate (NADPH).
Identified ChAT-positive neurons in the light microscope were subsequently studied in the electron microscope. All cholinergic neurons in the SN share essentially the same ultrastructural characteristics. The copious cytoplasm was rich in organelles with large lipofuscin granules. The synaptic input onto cell bodies and their dendrites was studied in serial sections. Synaptic contacts onto the perikarya and proximal dendrites were sparse and of asymmetric type. Both symmetric and asymmetric synaptic specializations onto ChAT-positive distal dendrites were detected. Asymmetric synaptic contacts onto cell bodies and dendrites were often defined by the presence of subjunctional dense bodies associated with the postsynaptic membrane. The pattern of the synaptic input to these cells differs strikingly from that onto unlabelled neighboring neurons. The perikarya and dendrites of the latter were characteristically covered with synaptic boutons.
Scarce immunoreactive terminals in asymmetric synaptic contact with unlabelled dendritic profiles were also detected in portions of SN compacta with no ChAT-positive cells.
Extranigrally located ChAT-positive cells of the PMT cholinergic complex were also examined in the electron microscope for comparison purposes. These cells exhibited, on the basis of their morphology and synaptic input pattern, very similar characteristics to those shown by SN cholinergic neurons.
On the basis of histochemical and morphological comparison between nigral cholinergic neurons and extranigrally located ChAT-positive cells of the PMT complex, it is concluded that cholinergic neurons in the SN of the rat may be projection neurons of the PMT cholinergic complex ectopically located and that they might play an important role in the regulation of forebrain activation and in locomotion. Our electron microscopic results suggest that substance P and GABA may influence the function of these cells.
Lamellar bodies are composed of stacks of closely-packed, ribosome-free cisterns which are in continuity with the rough endoplasmic reticulum. In the ferret nucleus basalis stained for choline acetyltransferase it was shown, by correlating light with electron microscopy, that only the cholinergic cells there possess lamellar bodies. The significance of lamellar bodies in the cholinergic neurons of the nucleus basalis is not known, but these structures may reflect a peculiar aspect of the functioning of the cholinergic cells which will need to be investigated further.
The laterodorsal tegmental nucleus (ntdl) contains a cluster of cells located just medial to the locus coeruleus in the pontine brainstem. The ntdl has been shown to project both rostrally to the forebrain and diencephalon and caudally to the spinal cord. In an effort to characterize this region neurochemically, the present study was conducted to identify a variety of neurochemicals localized within perikarya and fibers of the ntdl and surrounding nuclei. Rats were perfused with formalin, and brain sections were processed for fluorescence immunocytochemistry and acetylcholinesterase (AChE). Of the neurochemicals screened, atrial natriuretic factor (ANF), choline acetyltransferase (ChAT), cholecystokinin (CCK), calcitonin gene-related peptide (CGRP), dynorphin B (Dyn B), galanin, somatostatin, substance P, neurotensin (NT), neuropeptide Y (NPY), vasopressin, vasoactive intestinal polypeptide (VIP), serotonin (5HT), glutamic acid decarboxylase (GAD), and tyrosine hydroxylase (TH) were studied. AChE and ChAT staining revealed that the ntdl contains mostly cholinergic neurons. In addition, brightly reactive substance P and galanin and paler staining CRF, ANF, CGRP, NT, VIP, and Dyn B cell bodies were found within the ntdl. Varicose fibers in this nucleus also contained these peptides in addition to CCK, GAD, TH, 5HT, and NPY. The dorsal tegmental nucleus, dorsal raphe nucleus, locus coeruleus, and the parabrachial region contained a dense and varied assortment of peptides with distinct positions and patterns. This multiplicity of neurochemicals within this area suggests a possible influence on a variety of functions modulated by the ntdl and other closely associated tegmental nuclei.
The synaptic organization of the feline pedunculopontine tegmental nucleus (PPN) was studied electron microscopically. The bouton covering ratios were calculated in various sizes of PPN neurons, and the ratios of large neurons (56%) were found to be much higher than those of small neurons (16%). The PPN neuron dendrites usually showed some varicosities, and spines were observed on both somatic and dendritic profiles. Among a total of 1021 synapses sampled at random, axosomatic, axodendritic and axospinous synapses comprised 21.7, 61.2 and 14.1%, respectively. On the basis of the postsynaptic junction, these synapses were classified into the symmetric (66.3%) and the asymmetric (33.7%) types. The percentage of symmetric synapses was much higher on the soma (91.0%), and the large (69.4%) and medium-sized (63.2%) dendrite, while that of asymmetric synapses showed a higher value on the small dendrite (55.5%) and the dendritic spine (50.8%). Axoaxonic, dendrodendritic and dendroaxonic synapses, although not so frequent, were, in part, involved in the serial synapse or the synaptic triad. It is concluded that some PPN neurons are spiny, and that axosomatic, axodendritic and axospinous synapses are the main synaptic constituents and besides those synapses a more complex synaptic organization exists in this nucleus.
This investigation was carried out on the distribution of enkephalin-containing nerve fibres and terminals in the region of the nucleus basalis magnocellularis (NBM) of the rat. At the light microscope (LM) level, enkephalin-immunoreactive sites and endogenous choline acetyltransferase (ChAT) were demonstrated by employing the two-colour immunoperoxidase staining technique, using highly specific monoclonal antibodies against enkephalin and ChAT. A pharmacohistochemical procedure to reveal acetylcholinesterase (AChE)-synthesizing neurons combined with the peroxidase-antiperoxidase (PAP) immunocytochemical technique to detect endogenous enkephalins, provided ultrastructural data on the relationships of neuronal elements containing AChE and enkephalins in the region of the NBM.
At the LM level, cholinergic neurons of the NBM were surrounded by a dense network of enkephalin-immunoreactive nerve fibres. Electron microscopic (EM) observations of histochemically characterized structures, that were first identified in the LM, revealed that intensely AChE-stained structures in the region of the NBM received sparse synaptic inputs from enkephalin immunoreactive terminals. Synaptic inputs of immunoreactive terminals onto intensely AChE-stained neuron cell bodies were not detected. Synaptic contacts onto proximal AChE-positive dendrites were sparse, but the density increased on more distal regions of the dendrites. All immunoreactive boutons studied established symmetrical synaptic contacts with AChE-positive post-synaptic structures. The pattern of the synaptic input to these cells differs strikingly from that onto typical globus pallidus neurons. The perikarya and dendrites of the latter neurons were characteristically ensheathed in immunoreactive synaptic boutons.
Results are consistent with the view that enkephalin-like substances in the rat might be synaptic transmitters or neuromodulators in the area of the NBM and that cholinergic neurons of the NBM (Ch4) are integrated into the circuitry of the basal ganglia. Enkephalins may play an important role regulating the extrinsic cholinergic innervation of the neocortex.
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 mesencephalic locomotor region (MLR) was identified physiologically by inducing controlled locomotion on a treadmill in the precollicular rat following application of low amplitude current pulses to areas of the pontomesencephalic tegmentum. The same brains were processed using either of two techniques known to label neurons of the pedunculopontine nucleus (PPN)-choline acetyltransferase (ChAT) immunocytochemistry or nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Histological reconstruction of locomotion-inducing sites were localized within or adjacent to ChAT or NADPH-diaphorase labeled cell groups. Three dimensional reconstructions of the PPN were used to visualize the colocalization of low threshold locomotion-inducing stimulation sites within PPN neuronal aggregates. These findings lend further support to the suggestion that the PPN is part of the MLR. A theoretical framework is proposed to account for results derived from various lines of research on this area.