Lesioning of the pedunculopontine nucleus reduces rapid eye movement sleep, but does not alter cardiorespiratory activities during sleep, under hypoxic conditions in rats.

To determine how partial lesioning of the pedunculopontine nucleus (PPT) affects sleep, breathing, and blood pressure in rats, ibotenic acid (IBO) was injected bilaterally into the PPT. Sham-injected (saline) and IBO-lesioned rats were first studied under normoxic conditions (40 recordings were obtained from 15 rats, with each recording lasting for 6 daytime hours). Rats were then exposed to intermittent hypoxia for 4 ± 2 days (51 recordings from 12 rats, each lasting 6 daytime hours). The intermittent hypoxia protocol involved an oxygen decline lasting 35 s (to a nadir of 10 %) followed by a 50 s increase to normoxia. The IBO caused an estimated 53 % reduction in PPT neurons. When normoxic, IBO-lesioned rats had remarkedly normal sleep architecture, respiratory rates, and mean arterial pressure. The exposure to intermittent hypoxia evoked tachypnea in both the IBO-lesioned and sham-injected rats. When intermittently hypoxic, IBO-lesioned rats demonstrated a significant reduction in the duration of rapid eye movement (REM) sleep. We conclude that partial lesions of the PPT do not disrupt cardiorespiratory activities, but a reduction in PPT neurons impairs the ability to sustain REM sleep under hypoxic conditions.

Cholinergic Projections From the Pedunculopontine Tegmental Nucleus Contact Excitatory and Inhibitory Neurons in the Inferior Colliculus.

The inferior colliculus processes nearly all ascending auditory information. Most collicular cells respond to sound, and for a majority of these cells, the responses can be modulated by acetylcholine (ACh). The cholinergic effects are varied and, for the most part, the underlying mechanisms are unknown. The major source of cholinergic input to the inferior colliculus is the pedunculopontine tegmental nucleus (PPT), part of the pontomesencephalic tegmentum known for projections to the thalamus and roles in arousal and the sleep-wake cycle. Characterization of PPT inputs to the inferior colliculus has been complicated by the mixed neurotransmitter population within the PPT. Using selective viral-tract tracing techniques in a ChAT-Cre Long Evans rat, the present study characterizes the distribution and targets of cholinergic projections from PPT to the inferior colliculus. Following the deposit of viral vector in one PPT, cholinergic axons studded with boutons were present bilaterally in the inferior colliculus, with the greater density of axons and boutons ipsilateral to the injection site. On both sides, cholinergic axons were present throughout the inferior colliculus, distributing boutons to the central nucleus, lateral cortex, and dorsal cortex. In each inferior colliculus (IC) subdivision, the cholinergic PPT axons appear to contact both GABAergic and glutamatergic neurons. These findings suggest cholinergic projections from the PPT have a widespread influence over the IC, likely affecting many aspects of midbrain auditory processing. Moreover, the effects are likely to be mediated by direct cholinergic actions on both excitatory and inhibitory circuits in the inferior colliculus.

Morphological and electrophysiological properties of serotonin neurons with NMDA modulation in the mesencephalic locomotor region of neonatal ePet-EYFP mice.

The mesencephalic locomotor region (MLR) is an essential area for initiation of locomotion. Its functional roles and circuits underlying locomotion have been studied intensively in many species. Studies suggest that cuneiform nucleus and pedunculopontine nucleus (PPN) are two core regions in the MLR for locomotion. However, it remains unclear about cellular components and morphological and intrinsic membrane properties of the neurons in these regions, especially the serotonergic neurons. Using neonatal ePet-EYFP transgenic mice and immunofluorescent technique, we demonstrated existence of 5-HT neurons in the MLR and discovered that 5-HT neurons distributed mainly in the caudal PPN. 5-HT neurons were heterogeneous in MLR and had three types of firing pattern (single spike, phasic and tonic) and two subtypes of morphology (pyramidal and stellate). We measured parameters of 5-HT neurons (n = 35) including resting membrane potential (- 69.2 ± 4.2 mV), input resistance (1410.1 ± 616.9 MΩ), membrane capacitance (36.4 ± 14.9 pF), time constant (49.7 ± 19.4 ms), voltage threshold (- 32.1 ± 7.4 mV), rheobase (21.3 ± 12.4 pA), action potential amplitude (58.9 ± 12.8 mV) and half-width (4.7 ± 1.1 ms), afterhyperpolarization amplitude (23.6 ± 10.4 mV) and half-decay (331.6 ± 157.7 ms). 5-HT neurons were intrinsically different from adjacent non-5-HT neurons and less excitable than them. Hyperpolarization-activated inward currents and persistent inward currents were recorded in 5-HT neurons. NMDA increased excitability of 5-HT neurons, especially the tonic-firing neurons, accompanied with depolarization of membrane potential, hyperpolarization of voltage threshold, reduction of afterhyperpolarization half-decay, and left-shift of frequency-current relationship. This study provided insight into the distribution and properties of 5-HT neurons in the MLR and interaction between serotonergic and glutamatergic modulations.

Alterations in NMDAR-mediated signaling within the laterodorsal tegmental nucleus are associated with prenatal nicotine exposure.

Cigarette smoking during pregnancy has been clinically associated with a variety of poorbehavioral outcomes for the exposed individuals, including higher risks for drug abuse and development of attention/deficit-hyperactive disorder (ADHD). Experimental studies support the hypothesis that nicotine might contribute to these risks, since prenatal nicotine exposure (PNE) in rodents was associated with greater addiction liability, hyperactivity, social impairments and a wide range of emotional and cognitive deficits. Alterations of glutamate signaling within brain regions involved in behavioral circuits could contribute to these outcomes. The pontine laterodorsal tegmental nucleus (LDT) exerts cholinergic modulation within the ventral tegmental area, nucleus accumbens, and cortical-projecting thalamic centers and PNE-associated alterations in LDT glutamate signaling could impact cholinergic output to these LDT targets. We have previously demonstrated that PNE alters AMPA-mediated signaling within LDT neurons, and in the present investigation, we focused on changes of NMDA receptors (NMDARs) and presence of silent synapses as an indicator of metaplastic processes in LDT cells associated with PNE treatment. PNE was associated with a decreased functional presence of GluN2B NMDAR subunits in synapses of large, putatively cholinergic neurons, whereas an increased function of this subunit was detected in small, likely GABAergic cells. In addition, PNE was associated with functional alterations of extrasynaptic NMDARs in putative cholinergic neurons, suggestive of an increased presence of GluN3A-containing NMDARs. An increased number of silent synapses was exclusively seen in the small cells. When taken together, we hypothesize that NMDA-mediated signaling changes within LDT neurons following PNE treatment would result in reductions of excitatory cholinergic modulatory tone in target brain regions, which would be expected to contribute to the behavioral deficits found among these individuals.

Pedunculopontine Glutamatergic Neurons Provide a Novel Source of Feedforward Inhibition in the Striatum by Selectively Targeting Interneurons.

The main excitatory inputs to the striatum arising from the cortex and the thalamus innervate both striatal spiny projection neurons and interneurons. These glutamatergic inputs to striatal GABAergic interneurons have been suggested to regulate the spike timing of striatal projection neurons via feedforward inhibition. Understanding how different excitatory inputs are integrated within the striatal circuitry and how they regulate striatal output is crucial for understanding basal ganglia function and related behaviors. Here, using VGLUT2 mice from both sexes, we report the existence of a glutamatergic projection from the mesencephalic locomotor region to the striatum that avoids the spiny neurons and selectively innervates interneurons. Specifically, optogenetic activation of glutamatergic axons from the pedunculopontine nucleus induced monosynaptic excitation in most recorded striatal cholinergic interneurons and GABAergic fast-spiking interneurons. Optogenetic stimulation in awake head-fixed mice consistently induced an increase in the firing rate of putative cholinergic interneurons and fast-spiking interneurons. In contrast, this stimulation did not induce excitatory responses in spiny neurons but rather disynaptic inhibitory responses ex vivo and a decrease in their firing rate in vivo, suggesting a feedforward mechanism mediating the inhibition of spiny projection neurons through the selective activation of striatal interneurons. Furthermore, unilateral stimulation of pedunculopontine nucleus glutamatergic axons in the striatum induced ipsilateral head rotations consistent with the inhibition of striatal output neurons. Our results demonstrate the existence of a unique interneuron-specific midbrain glutamatergic input to the striatum that exclusively recruits feedforward inhibition mechanisms.SIGNIFICANCE STATEMENT Glutamatergic inputs to the striatum have been shown to target both striatal projection neurons and interneurons and have been proposed to regulate spike timing of the projection neurons in part through feedforward inhibition. Here, we reveal the existence of a midbrain source of glutamatergic innervation to the striatum, originating in the pedunculopontine nucleus. Remarkably, this novel input selectively targets striatal interneurons, avoiding the projection neurons. Furthermore, we show that this selective innervation of interneurons can regulate the firing of the spiny projection neurons and inhibit the striatal output via feedforward inhibition. Together, our results describe a unique source of excitatory innervation to the striatum which selectively recruits feedforward inhibition of spiny neurons without any accompanying excitation.

Class II histone deacetylases require P/Q-type Ca2+ channels and CaMKII to maintain gamma oscillations in the pedunculopontine nucleus.

Epigenetic mechanisms (i.e., histone post-translational modification and DNA methylation) play a role in regulation of gene expression. The pedunculopontine nucleus (PPN), part of the reticular activating system, manifests intrinsic gamma oscillations generated by voltage-dependent, high threshold N- and P/Q-type Ca2+ channels. We studied whether PPN intrinsic gamma oscillations are affected by inhibition of histone deacetylation. We showed that, a) acute in vitro exposure to the histone deacetylation Class I and II inhibitor trichostatin A (TSA, 1 µM) eliminated oscillations in the gamma range, but not lower frequencies, b) pre-incubation with TSA (1 µM, 90-120 min) also decreased gamma oscillations, c) Ca2+ currents (ICa) were reduced by TSA, especially on cells with P/Q-type channels, d) a HDAC Class I inhibitor MS275 (500 nM), and a Class IIb inhibitor Tubastatin A (150-500 nM), failed to affect gamma oscillations, e) MC1568, a HDAC Class IIa inhibitor (1 µM), blocked gamma oscillations, and f) the effects of both TSA and MC1568 were blunted by blockade of CaMKII with KN-93 (1 µM). These results suggest a cell type specific effect on gamma oscillations when histone deacetylation is blocked, suggesting that gamma oscillations through P/Q-type channels modulated by CaMKII may be linked to processes related to gene transcription.

Parallel descending dopaminergic connectivity of A13 cells to the brainstem locomotor centers.

The mesencephalic locomotor region (MLR) is an important integrative area for the initiation and modulation of locomotion. Recently it has been realized that dopamine (DA) projections from the substantia nigra pars compacta project to the MLR. Here we explore DA projections from an area of the medial zona incerta (ZI) known for its role in motor control onto the MLR. We provide evidence that dopaminergic (DAergic) A13 neurons have connectivity to the cuneiform nucleus (CnF) and pedunculopontine tegmental nucleus (PPTg) of the MLR. No ascending connectivity to the dorsolateral striatum was observed. On the other hand, DAergic A13 projections to the medullary reticular formation (MRF) and the lumbar spinal cord were sparse. A small number of non-DAergic neurons within the medial ZI projected to the lumbar spinal cord. We then characterized the DA A13 cells and report that these cells differ from canonical DA neurons since they lack the Dopamine Transporter (DAT). The lack of DAT expression, and possibly the lack of a dopamine reuptake mechanism, points to a longer time of action compared to typical dopamine neurons. Collectively our data suggest a parallel descending DAergic pathway from the A13 neurons of the medial ZI to the MLR, which we expect is important for modulating movement.

Cocaine Selectively Reorganizes Excitatory Inputs to Substantia Nigra Pars Compacta Dopamine Neurons.

Substantia nigra pars compacta (SNc) dopamine neurons and their targets are involved in addiction and cue-induced relapse. However, afferents onto SNc dopamine neurons themselves appear insensitive to drugs of abuse, such as cocaine, when afferents are collectively stimulated electrically. This contrasts with ventral tegmental area (VTA) dopamine neurons, whose glutamate afferents react robustly to cocaine. We used an optogenetic strategy to isolate identified SNc inputs and determine whether cocaine sensitivity in the mouse SNc circuit is conferred at the level of three glutamate afferents: dorsal raphé nucleus (DR), pedunculopontine nucleus (PPN), and subthalamic nucleus (STN). We found that excitatory afferents to SNc dopamine neurons are sensitive to cocaine in an afferent-specific manner. A single exposure to cocaine in vivo led to PPN-innervated synapses reducing the AMPA-to-NMDA receptor-mediated current ratio. In contrast to work in the VTA, this was due to increased NMDA receptor function with no change in AMPA receptor function. STN synapses showed a decrease in calcium-permeable AMPA receptors after cocaine, but no change in the AMPA-to-NMDA ratio. Cocaine also increased the release probability at DR-innervated and STN-innervated synapses, quantified by decreases in paired-pulse ratios. However, release probability at PPN-innervated synapses remained unaffected. By examining identified inputs, our results demonstrate a functional distribution among excitatory SNc afferent nuclei in response to cocaine, and suggest a compelling architecture for differentiation and separate parsing of inputs within the nigrostriatal system.SIGNIFICANCE STATEMENT Prior studies have established that substantia nigra pars compacta (SNc) dopamine neurons are a key node in the circuitry that drives addiction and relapse, yet cocaine apparently has no effect on electrically stimulated excitatory inputs. Our study is the first to demonstrate the functional impact of a drug of abuse on synaptic mechanisms of identified afferents to the SNc. Optogenetic dissection of inputs originating from dorsal raphé, pedunculopontine, and subthalamic nuclei were tested for synaptic modifications following in vivo cocaine exposure. Our results demonstrate that cocaine differentially induces modifications to SNc synapses depending on input origin. This presents implications for understanding dopamine processing of motivated behavior; most critically, it indicates that dopamine neurons selectively modulate signal reception processed by afferent nuclei.

Cholinergic Projections to the Substantia Nigra Pars Reticulata Inhibit Dopamine Modulation of Basal Ganglia through the M4 Muscarinic Receptor.

Cholinergic regulation of dopaminergic inputs into the striatum is critical for normal basal ganglia (BG) function. This regulation of BG function is thought to be primarily mediated by acetylcholine released from cholinergic interneurons (ChIs) acting locally in the striatum. We now report a combination of pharmacological, electrophysiological, optogenetic, chemogenetic, and functional magnetic resonance imaging studies suggesting extra-striatal cholinergic projections from the pedunculopontine nucleus to the substantia nigra pars reticulata (SNr) act on muscarinic acetylcholine receptor subtype 4 (M4) to oppose cAMP-dependent dopamine receptor subtype 1 (D1) signaling in presynaptic terminals of direct pathway striatal spiny projections neurons. This induces a tonic inhibition of transmission at direct pathway synapses and D1-mediated activation of motor activity. These studies provide important new insights into the unique role of M4 in regulating BG function and challenge the prevailing hypothesis of the centrality of striatal ChIs in opposing dopamine regulation of BG output.

Interaction between neuronal calcium sensor protein 1 and lithium in pedunculopontine neurons.

Bipolar disorder is characterized by sleep dysregulation, suggesting a role for the reticular activating system (RAS). Postmortem studies showed increased expression of neuronal calcium sensor protein 1 (NCS-1) in the brains of some bipolar disorder patients, and reduced or aberrant gamma band activity is present in the same disorder. Lithium (Li+) has been shown to effectively treat the mood disturbances in bipolar disorder patients. We previously showed that NCS-1 at low levels increased, and at high levels decreased, gamma oscillations in RAS pedunculopontine neurons (PPN), and that Li+ decreased these oscillations. We previously described the effects of each agent on oscillations, G-protein mechanisms, and Ca2+ currents. However, we designed the present experiments to determine the nature of the interaction of NCS-1 and Li+ at physiological concentrations that would have an effect within minutes of application. As expected, Li+ decreased gamma oscillation amplitude, while NCS-1 increased the amplitude of gamma oscillations. We identified NCS-1 at 2 µmol/L as a concentration that increased gamma oscillations within 5-10 min, and Li+ at 10 µmol/L as a concentration that decreased gamma oscillations within 5 min. The combined application of NCS-1 and Li+ at these concentrations showed that Li+ reduced the effects of NCS-1 on oscillation amplitude within 5-10 min. These results demonstrate that at physiological levels, Li+ acts to reduce the effects of NCS-1 so that, given over expression of NCS-1, Li+ would have salutary effects.

Rethinking the Pedunculopontine Nucleus: From Cellular Organization to Function.

The pedunculopontine nucleus (PPN) has long been considered an interface between the basal ganglia and motor systems, and its ability to regulate arousal states puts the PPN in a key position to modulate behavior. Despite the large amount of data obtained over recent decades, a unified theory of its function is still incomplete. By putting together classical concepts and new evidence that dissects the influence of its different neuronal subtypes on their various targets, we propose that the PPN and, in particular, cholinergic neurons have a central role in updating the behavioral state as a result of changes in environmental contingencies. Such a function is accomplished by a combined mechanism that simultaneously restrains ongoing obsolete actions while it facilitates new contextual associations.

Effects of bilateral vestibular deafferentation in rat on hippocampal theta response to somatosensory stimulation, acetylcholine release, and cholinergic neurons in the pedunculopontine tegmental nucleus.

Vestibular dysfunction has been shown to cause spatial memory impairment. Neurophysiological studies indicate that bilateral vestibular loss (BVL), in particular, is associated with an impairment of the response of hippocampal place cells and theta rhythm. However, the specific neural pathways through which vestibular information reaches the hippocampus are yet to be fully elucidated. The aim of the present study was to further investigate the hypothesised 'theta-generating pathway' from the brainstem vestibular nucleus to the hippocampus. BVL, and in some cases, unilateral vestibular loss (UVL), induced by intratympanic sodium arsanilate injections in rats, were used to investigate the effects of vestibular loss on somatosensory-induced type 2 theta rhythm, acetylcholine (ACh) release in the hippocampus, and the number of cholinergic neurons in the pedunculopontine tegmental nucleus (PPTg), an important part of the theta-generating pathway. Under urethane anaesthesia, BVL was found to cause a significant increase in the maximum power of the type 2 theta (3-6 Hz) frequency band compared to UVL and sham animals. Rats with BVL generally exhibited a lower basal level of ACh release than sham rats; however, this difference was not statistically significant. The PPTg of BVL rats exhibited significantly more choline-acetyltransferase (ChAT)-positive neurons than that of sham animals, as did the contralateral PPTg of UVL animals; however, the number of ChAT-positive neurons on the ipsilateral side of UVL animals was not significantly different from sham animals. The results of these studies indicate that parts of the theta-generating pathway undergo a significant reorganisation following vestibular loss, which suggests that this pathway is important for the interaction between the vestibular system and the hippocampus.

Activation of Pedunculopontine Glutamate Neurons Is Reinforcing.

Dopamine transmission from midbrain ventral tegmental area (VTA) neurons underlies behavioral processes related to motivation and drug addiction. The pedunculopontine tegmental nucleus (PPTg) is a brainstem nucleus containing glutamate-, acetylcholine-, and GABA-releasing neurons with connections to basal ganglia and limbic brain regions. Here we investigated the role of PPTg glutamate neurons in reinforcement, with an emphasis on their projections to VTA dopamine neurons. We used cell-type-specific anterograde tracing and optogenetic methods to selectively label and manipulate glutamate projections from PPTg neurons in mice. We used anatomical, electrophysiological, and behavioral assays to determine their patterns of connectivity and ascribe functional roles in reinforcement. We found that photoactivation of PPTg glutamate cell bodies could serve as a direct positive reinforcer on intracranial self-photostimulation assays. Further, PPTg glutamate neurons directly innervate VTA; photostimulation of this pathway preferentially excites VTA dopamine neurons and is sufficient to induce behavioral reinforcement. These results demonstrate that ascending PPTg glutamate projections can drive motivated behavior, and PPTg to VTA synapses may represent an important target relevant to drug addiction and other mental health disorders. SIGNIFICANCE STATEMENT: Uncovering brain circuits underlying reward-seeking is an important step toward understanding the circuit bases of drug addiction and other psychiatric disorders. The dopaminergic system emanating from the ventral tegmental area (VTA) plays a key role in regulating reward-seeking behaviors. We used optogenetics to demonstrate that the pedunculopontine tegmental nucleus sends glutamatergic projections to VTA dopamine neurons, and that stimulation of this circuit promotes behavioral reinforcement. The findings support a critical role for pedunculopontine tegmental nucleus glutamate neurotransmission in modulating VTA dopamine neuron activity and behavioral reinforcement.

ExPPNing how acetylcholine improves gait in Parkinson's disease: An Editorial Highlight for 'Deletion of the Vesicular Acetylcholine Transporter from Pedunculopontine/laterodorsal tegmental neurons modifies gait'.

Read the highlighted article 'Deletion of the Vesicular Acetylcholine Transporter from Pedunculopontine/laterodorsal tegmental neurons modifies gait' on page 787.

Cholinergic, Glutamatergic, and GABAergic Neurons of the Pedunculopontine Tegmental Nucleus Have Distinct Effects on Sleep/Wake Behavior in Mice.

The pedunculopontine tegmental (PPT) nucleus has long been implicated in the regulation of cortical activity and behavioral states, including rapid eye-movement (REM) sleep. For example, electrical stimulation of the PPT region during sleep leads to rapid awakening, whereas lesions of the PPT in cats reduce REM sleep. Though these effects have been linked with the activity of cholinergic PPT neurons, the PPT also includes intermingled glutamatergic and GABAergic cell populations, and the precise roles of cholinergic, glutamatergic, and GABAergic PPT cell groups in regulating cortical activity and behavioral state remain unknown. Using a chemogenetic approach in three Cre-driver mouse lines, we found that selective activation of glutamatergic PPT neurons induced prolonged cortical activation and behavioral wakefulness, whereas inhibition reduced wakefulness and increased non-REM (NREM) sleep. Activation of cholinergic PPT neurons suppressed lower-frequency electroencephalogram rhythms during NREM sleep. Last, activation of GABAergic PPT neurons slightly reduced REM sleep. These findings reveal that glutamatergic, cholinergic, and GABAergic PPT neurons differentially influence cortical activity and sleep/wake states. SIGNIFICANCE STATEMENT: More than 40 million Americans suffer from chronic sleep disruption, and the development of effective treatments requires a more detailed understanding of the neuronal mechanisms controlling sleep and arousal. The pedunculopontine tegmental (PPT) nucleus has long been considered a key site for regulating wakefulness and REM sleep. This is mainly because of the cholinergic neurons contained in the PPT nucleus. However, the PPT nucleus also contains glutamatergic and GABAergic neurons that likely contribute to the regulation of cortical activity and sleep-wake states. The chemogenetic experiments in the present study reveal that cholinergic, glutamatergic, and GABAergic PPT neurons each have distinct effects on sleep/wake behavior, improving our understanding of how the PPT nucleus regulates cortical activity and behavioral states.

Deletion of the vesicular acetylcholine transporter from pedunculopontine/laterodorsal tegmental neurons modifies gait.

Postural instability and gait disturbances, common disabilities in the elderly and frequently present in Parkinson's disease (PD), have been suggested to be related to dysfunctional cholinergic signaling in the brainstem. We investigated how long-term loss of cholinergic signaling from mesopontine nuclei influence motor behaviors. We selectively eliminated the vesicular acetylcholine transporter (VAChT) in pedunculopontine and laterodorsal tegmental nuclei cholinergic neurons to generate mice with selective mesopontine cholinergic deficiency (VAChTEn1-Cre-flox/flox ). VAChTEn1-Cre-flox/flox mice did not show any gross health or neuromuscular abnormality on metabolic cages, wire-hang and grip-force tests. Young VAChTEn1-Cre-flox/flox mice (2-5 months-old) presented motor learning/coordination deficits on the rotarod; moved slower, and had smaller steps on the catwalk, but showed no difference in locomotor activity on the open field. Old VAChTEn1-Creflox/flox mice (13-16 months-old) showed more pronounced motor learning/balance deficits on the rotarod, and more pronounced balance deficits on the catwalk. Furthermore, old mutants moved faster than controls, but with similar step length. Additionally, old VAChT-deficient mice were hyperactive. These results suggest that dysfunction of cholinergic neurons from mesopontine nuclei, which is commonly seen in PD, has causal roles in motor functions. Prevention of mesopontine cholinergic failure may help to prevent/improve postural instability and falls in PD patients. Read the Editorial Highlight for this article on page 688.

Cholinergic and glutamatergic transmission at synapses between pedunculopotine tegmental nucleus axonal terminals and A7 catecholamine cell group noradrenergic neurons in the rat.

We characterized transmission from the pedunculopotine tegmental nucleus (PPTg), which contains cholinergic and glutamatergic neurons, at synapses with noradrenergic (NAergic) A7 neurons. Injection of an anterograde neuronal tracer, biotinylated-dextran amine, into the PPTg resulted in labeling of axonal terminals making synaptic connection with NAergic A7 neurons. Consistent with this, extracellular stimulation using a train of 10 pulses at 100 Hz evoked both fast and slow excitatory synaptic currents (EPSCs) that were blocked, respectively, by DNQX, a non-N-methyl-d-aspartate receptor blocker, or atropine, a cholinergic muscarinic receptor (mAChR) blocker. Interestingly, many spontaneous-like, but stimulation-dependent, EPSCs, were seen for up to one second after the end of stimulation and were blocked by DNQX and decreased by EGTA-AM, a membrane permeable form of EGTA, showing they are glutamatergic EPSCs causing by asynchronous release of vesicular quanta. Moreover, application of atropine or carbachol, an mAChR agonist, caused, respectively, an increase in the number of asynchronous EPSCs or a decrease in the frequency of miniature EPSCs, showing that mAChRs mediated presynaptic inhibition of glutamatergic transmission of the PPTg onto NAergic A7 neurons. In conclusion, our data show direct synaptic transmission of PPTg afferents onto pontine NAergic neurons that involves cooperation of cholinergic and glutamatergic transmission. This dual-transmitter transmission drives the firing rate of NAergic neurons, which may correlate with axonal and somatic/dendritic release of NA.

Intracellular mechanisms modulating gamma band activity in the pedunculopontine nucleus (PPN).

The pedunculopontine nucleus is a part of the reticular activating system, and is active during waking and REM sleep. Previous results showed that all PPN cells tested fired maximally at gamma frequencies when depolarized. This intrinsic membrane property was shown to be mediated by high-threshold N- and P/Q-type Ca(2+) channels. Recent studies show that the PPN contains three independent populations of neurons which can generate gamma band oscillations through only N-type channels, only P/Q-type channels, or both N- and P/Q-type channels. This study investigated the intracellular mechanisms modulating gamma band activity in each population of neurons. We performed in vitro patch-clamp recordings of PPN neurons from Sprague-Dawley rat pups, and applied 1-sec ramps to induce intrinsic membrane oscillations. Our results show that there are two pathways modulating gamma band activity in PPN neurons. We describe populations of neurons mediating gamma band activity through only N-type channels and the cAMP/PKA pathway (presumed "REM-on" neurons), through only P/Q-type channels and the CaMKII pathway (presumed "Wake-on" neurons), and a third population which can mediate gamma activity through both N-type channels and cAMP/PK and P/Q-type channels and CaMKII (presumed "Wake/REM-on" neurons). These novel results suggest that PPN gamma oscillations are modulated by two independent pathways related to different Ca(2+) channel types.

On the Role of the Pedunculopontine Nucleus and Mesencephalic Reticular Formation in Locomotion in Nonhuman Primates.

UNLABELLED: The mesencephalic reticular formation (MRF) is formed by the pedunculopontine and cuneiform nuclei, two neuronal structures thought to be key elements in the supraspinal control of locomotion, muscle tone, waking, and REM sleep. The role of MRF has also been advocated in modulation of state of arousal leading to transition from wakefulness to sleep and it is further considered to be a main player in the pathophysiology of gait disorders seen in Parkinson's disease. However, the existence of a mesencephalic locomotor region and of an arousal center has not yet been demonstrated in primates. Here, we provide the first extensive electrophysiological mapping of the MRF using extracellular recordings at rest and during locomotion in a nonhuman primate (NHP) (Macaca fascicularis) model of bipedal locomotion. We found different neuronal populations that discharged according to a phasic or a tonic mode in response to locomotion, supporting the existence of a locomotor neuronal circuit within these MRF in behaving primates. Altogether, these data constitute the first electrophysiological characterization of a locomotor neuronal system present within the MRF in behaving NHPs under normal conditions, in accordance with several studies done in different experimental animal models. SIGNIFICANCE STATEMENT: We provide the first extensive electrophysiological mapping of the two major components of the mesencephalic reticular formation (MRF), namely the pedunculopontine and cuneiform nuclei. We exploited a nonhuman primate (NHP) model of bipedal locomotion with extracellular recordings in behaving NHPs at rest and during locomotion. Different MRF neuronal groups were found to respond to locomotion, with phasic or tonic patterns of response. These data constitute the first electrophysiological evidences of a locomotor neuronal system within the MRF in behaving NHPs.

Cholinergic excitation from the pedunculopontine tegmental nucleus to the dentate nucleus in the rat.

In spite of the existence of pedunculopontine tegmental nucleus (PPTg) projections to cerebellar nuclei, their nature and functional role is unknown. These fibers may play a crucial role in postural control and may be involved in the beneficial effects induced by deep-brain stimulation (DBS) of brainstem structures in motor disorders. We investigated the effects of PPTg microstimulation on single-unit activity of dentate, fastigial and interpositus nuclei. The effects of PPTg stimulation were also studied in rats whose PPTg neurons were destroyed by ibotenic acid and subsequently subjected to iontophoretically applied cholinergic antagonists. The main response recorded in cerebellar nuclei was a short-latency (1.5-2 ms) and brief (13-15 ms) orthodromic activation. The dentate nucleus was the most responsive to PPTg stimulation. The destruction of PPTg cells reduced the occurrence of PPTg-evoked activation of dentate neurons, suggesting that the effect was due to stimulation of cell bodies and not due to fibers passing through or close to the PPTg. Application of cholinergic antagonists reduced or eliminated the PPTg-evoked response recorded in the dentate nucleus. The results show that excitation is exerted by the PPTg on the cerebellar nuclei, in particular on the dentate nucleus. Taken together with the reduction of nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in lesioned animals, the iontophoretic experiments suggest that the activation of dentate neurons is due to cholinergic fibers. These data help to explain the effects of DBS of the PPTg on axial motor disabilities in neurodegenerative disorders.