Connexin-36-positive gap junctions in ventral tegmental area GABA neurons sustain opiate dependence.

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can transform from a form of pleasure-seeking to a form of relief-seeking. Ventral tegmental area (VTA) GABA neurons form an anatomical point of divergence between two double dissociable pathways that have been shown to be functionally implicated and necessary for these respective motivations to seek drugs. The tegmental pedunculopontine nucleus (TPP) is necessary for opiate conditioned place preferences (CPP) in previously drug-naïve rats and mice, whereas dopaminergic (DA) transmission in the nucleus accumbens (NAc) is necessary for opiate CPP in opiate-dependent and withdrawn (ODW) rats and mice. Here, we show that this switch in functional anatomy is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve-like state wherein the TPP was necessary for opiate CPP and where opiate withdrawal aversions were lost. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36 fl(CFP)/fl(CFP)) exhibited a perpetual drug-naïve-like state wherein opiate CPP was always DA independent, and opiate withdrawal aversions were absent even in mice subjected to an opiate dependence and withdrawal induction protocol. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to an ODW state wherein opiate CPP was DA dependent. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.

Beta band modulation by dopamine D2 receptors in the primary motor cortex and pedunculopontine nucleus in a rat model of Parkinson's disease.

Beta band (12-30 Hz) hypersynchrony within the basal ganglia-thalamocortical network has been suggested as a hallmark of Parkinson's disease (PD) pathophysiology. Abnormal beta band oscillations are found in the pedunculopontine nucleus (PPN) and primary motor cortex (M1) and are correlated with dopamine depletion. Dopamine acts locomotion and motor performance mainly through dopamine receptors (D1 and D2). However, the precise mechanism by which dopamine receptors regulate beta band electrophysiological activities between the PPN and M1 is still unknown. Here, we recorded the neuronal activity of the PPN and M1 simultaneously by the administration of the drug (SCH23390 and raclopride), selectively blocking the dopamine D1 receptor and D2 receptor. We discovered that the increased coherent activity of the beta band (12-30 Hz) between M1 and PPN in the lesioned group could be reduced and restored by injecting raclopride in the resting and wheel running states. Our studies revealed the unique role of D2 dopamine receptor signaling in regulating ß band oscillatory activity in M1 and PPN and their relationship after the loss of dopamine, which contributes to elucidating the underlying mechanism of the pathophysiology of PD.

Levodopa affects spike and local field synchronisation in the pedunculopontine nucleus of a rat model of Parkinson's disease.

The pedunculopontine nucleus (PPN) undergoes significant anatomic and electrophysiological alterations in Parkinson's disease (PD), severely impacting locomotion. However, the effect of 6-hydroxydopamine (6-OHDA) lesion and levodopa (L-DOPA) therapy on the relationships between spike activities and local field potential (LFP) within the PPN is not well-understood. Synchronisation between the spike activity of individual neurones and LFP of neuronal ensembles is a crucial problem in the pathogenesis of PD. In this study, LFP signals and spikes in the PPN of rats in control, lesioned, and L-DOPA groups were recorded synchronously with a multi-unit electrical signal acquisition system and analysed for their coherence value, spike-field coherence, and phase-lock relationship. The spike-LFP relationship in the PPN was markedly increased in specific frequency bands because of the 6-OHDA lesion but differed depending on the animal locomotion state and neuronal type. L-DOPA had a limited therapeutic effect on the 6-OHDA-induced increase in the coherence value. Our study demonstrates that the PPN spike-LFP relationship is involved in the pathogenesis of PD and is critical for the effects of L-DOPA, providing a basis for the clinical treatment of refractory PD symptoms.

DREADD Activation of Pedunculopontine Cholinergic Neurons Reverses Motor Deficits and Restores Striatal Dopamine Signaling in Parkinsonian Rats.

The brainstem-based pedunculopontine nucleus (PPN) traditionally associates with motor function, but undergoes extensive degeneration during Parkinson's disease (PD), which correlates with axial motor deficits. PPN-deep brain stimulation (DBS) can alleviate certain symptoms, but its mechanism(s) of action remains unknown. We previously characterized rats hemi-intranigrally injected with the proteasomal inhibitor lactacystin, as an accurate preclinical model of PD. Here we used a combination of chemogenetics with positron emission tomography imaging for in vivo interrogation of discrete neural networks in this rat model of PD. Stimulation of excitatory designer receptors exclusively activated by designer drugs expressed within PPN cholinergic neurons activated residual nigrostriatal dopaminergic neurons to produce profound motor recovery, which correlated with striatal dopamine efflux as well as restored dopamine receptor 1- and dopamine receptor 2-based medium spiny neuron activity, as was ascertained with c-Fos-based immunohistochemistry and stereological cell counts. By revealing that the improved axial-related motor functions seen in PD patients receiving PPN-DBS may be due to stimulation of remaining PPN cholinergic neurons interacting with dopaminergic ones in both the substantia nigra pars compacta and the striatum, our data strongly favor the PPN cholinergic-midbrain dopaminergic connectome as mechanism for PPN-DBS's therapeutic effects. These findings have implications for refining PPN-DBS as a promising treatment modality available to PD patients.

Differential effects of HDAC inhibitors on PPN oscillatory activity in vivo.

The pedunculopontine nucleus (PPN) has long been known to be part of the reticular activating system (RAS) in charge of arousal and REM sleep. We previously showed that in vitro exposure to a HDAC Class I and II mixed inhibitor (TSA), or a specific HDAC class IIa inhibitor (MC 1568), decreased PPN gamma oscillations. Given the lack of information on systemic in vivo treatments on neuronal synaptic properties, the present study was designed to investigate the systemic effect of HDAC inhibitors (HDACi) on PPN rhythmicity. Rat pups were injected (acute, single dose) with TSA (4 or 20 mg/kg), MC1568 (4 or 20 mg/kg), or MS275 (20 or 100 mg/kg). Our results show that MC1568 (20 mg/kg) reduced mean frequency of PPN oscillations at gamma band, while increasing mean input resistance (Rm) of PPN neurons. For TSA (4 and 20 mg/kg), we observed reduced mean frequency of oscillations at gamma band and increased mean Rm of PPN neurons. Systemic administration of 20 mg/kg MC1568 and TSA effects on Rm were washed out after 60 min of in vitro incubation of PPN slices, suggesting an underlying functional recovery of PPN calcium-mediated gamma band oscillations over time. In addition, at a lower dose, 4 mg/kg, MC1568 and TSA induced higher mean amplitude gamma oscillations. Blocking HDAC class I might not have deleterious effects on gamma activity, but, more importantly, the inhibition of HDAC class I (at 100 mg/kg) increased gamma band oscillations. In conclusion, the present results in vivo validate our previous findings in vitro and further expand information on the effects of HDAC inhibition on PPN rhythmicity. PPN neurons require normal activity of HDAC class IIa in order to oscillate at gamma band.

Neuronal oscillations of the pedunculopontine nucleus in progressive supranuclear palsy: Influence of levodopa and movement.

OBJECTIVE: The pedunculopontine nucleus (PPN) has been proposed as a new deep brain stimulation (DBS) target for the treatment in idiopathic Parkinson's syndrome (IPS) and progressive supranuclear palsy (PSP). In IPS, levodopa has been shown to induce alpha activity in the PPN, indicating a possible physiological role for these oscillations in movement control. Despite shared clinical features, the PPN is more severely affected in PSP than IPS. Here we investigated neuronal oscillations in the PPN in PSP and the influence of levodopa and movement. METHODS: Local field potentials were recorded bilaterally from the PPN of 4 PSP patients at rest, with levodopa and during self-paced leg movements. RESULTS: During rest, levodopa administration was associated with significantly increased alpha and reduced gamma activity in the PPN. Without levodopa, continuous movements were associated with reduced alpha and beta power. These differences between oscillatory power during movement and resting state were not observed with levodopa administration. CONCLUSION: In PSP the changes in neuronal oscillations in the PPN region on levodopa administration are similar to those reported in IPS. The enhancement of lower frequency oscillations in the PPN is possibly influenced by a dopaminergic activation of the striatal pathway and a reduced pallidal inhibition. SIGNIFICANCE: Levodopa influences neuronal oscillations at low and high frequencies in the PPN region in Parkinsonian disorders.

Effects of chronic alcohol consumption and withdrawal on the cholinergic neurons of the pedunculopontine and laterodorsal tegmental nuclei of the rat: An unbiased stereological study.

The brain cholinergic system comprises two main recognized subdivisions, the basal forebrain and the brainstem cholinergic systems. The effects of chronic alcohol consumption on the basal forebrain cholinergic nuclei have been investigated extensively, but there is only one study that has examined those effects on the brainstem cholinergic nuclei. The last one comprises the pedunculopontine tegmental (PPT) and the laterodorsal tegmental (LDT) nuclei, which are known to give origin to the main cholinergic projection to the ventral tegmental area, a key brain region of the neural circuit, the mesocorticolimbic system, that mediates several behavioral and physiological processes, including reward. In the present study, we have examined, using stereological methods, the effects of chronic alcohol consumption (6 months) and subsequent withdrawal (2 months) on the total number and size of PPT and LDT choline acetyltransferase (ChAT)-immunoreactive neurons. The total number of PPT and LDT ChAT-immunoreactive neurons was unchanged in ethanol-treated and withdrawn rats. However, ChAT-immunoreactive neurons were significantly hypertrophied in ethanol-treated rats, an alteration that did not revert 2 months after ethanol withdrawal. These results show that prolonged exposure to ethanol leads to long-lasting, and potentially irreversible, cytoarchitectonic and neurochemical alterations in the brainstem cholinergic nuclei. These alterations suggest that the alcohol-induced changes in the brainstem cholinergic nuclei might play a role in the mechanisms underlying the development of addictive behavior to alcohol.

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.

Spike and Local Field Synchronization Between the Pedunculopontine Nucleus and Primary Motor Cortex in a Rat Model of Parkinson's Disease.

The pedunculopontine nucleus (PPN) shows altered electrophysiological and anatomic characteristics in Parkinson's disease (PD), but little is known about the effect of 6-hydroxydopamine (6-OHDA) lesion and levodopa (L-DOPA) therapy on the relationship between spike and local field potential (LFP) activities in the PPN and motor cortex. Aiming to investigate this, synchronous spike and LFP signals in the PPN and primary motor cortex (M1) were recorded. The spike-LFP relationship was evaluated using coherence analysis, phase-lock and spike-field coherence (SFC). The results suggested that 6-OHDA lesion had a significant effect on the spike-LFP relationship between the PPN and M1 in rats under a rest or locomotion state. The significantly altered frequency bands varied across different neuron types and animal activity states. In addition, the altered coherence values between PPN spike and M1 LFP were refractory to long-term L-DOPA therapy although all other changes could be reversed by this drug treatment. All results provided evidence of the spike-LFP relationship between the PPN and M1 in PD, revealing some network mechanisms of the cortico-basal ganglia circuitry and PPN, which might be an underlying candidate for PD pathophysiology and therapy.

The antinociceptive effect of anterior pretectal nucleus stimulation is mediated by distinct neurotransmitter mechanisms in descending pain pathways.

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.

Reciprocal interaction between monoaminergic systems and the pedunculopontine nucleus: Implication in the mechanism of L-DOPA.

The pedunculopontine nucleus (PPN) is part of the mesencephalic locomotor region (MLR) and has been involved in the control of gait, posture, locomotion, sleep, and arousal. It likely participates in some motor and non-motor symptoms of Parkinson's disease and is regularly proposed as a surgical target to ameliorate gait, posture and sleep disorders in Parkinsonian patients. The PPN overlaps with the monoaminergic systems including dopamine, serotonin and noradrenaline in the modulation of the above-mentioned functions. All these systems are involved in Parkinson's disease and the mechanism of the anti-Parkinsonian agents, mostly L-DOPA. This suggests that PPN interacts with monoaminergic neurons and vice versa. Some evidence indicates that the PPN sends cholinergic, glutamatergic and even gabaergic inputs to mesencephalic dopaminergic cells, with the data regarding serotonergic or noradrenergic cells being less well known. Similarly, the control exerted by the PPN on dopaminergic neurons, is multiple and complex, and more extensively explored than the other monoaminergic systems. The data on the influence of monoaminergic systems on PPN neuron activity are rather scarce. While there is evidence that the PPN influences the therapeutic response of L-DOPA, it is still difficult to discerne the reciprocal action of the PPN and monoaminergic systems in this action. Additional data are required to better understand the functional organization of monoaminergic inputs to the MLR including the PPN to get a clearer picture of their interaction.

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.

Brain sites mediating cyclosomatostatin-induced catalepsy in Wistar rats: A specific role for the nigrostriatal system and locus coeruleus.

A decrease in somatostatin activity is observed in the Parkinsonian brain. In recent experiments on rats, we simulated this abnormality by intracerebroventricular injections of a somatostatin antagonist, cyclosomatostatin. The treated animals displayed catalepsy, a state that resembles the extrapyramidal signs of Parkinson's disease. The neuroanatomical substrates mediating the catalepsy-inducing effect of cyclosomatostatin are unknown. To clarify this issue, we assessed here the action of cyclosomatostatin injected into the substantia nigra pars compacta (SNc), dorsal striatum (DS), locus coeruleus (LC), pedunculopontine tegmental nucleus (PPTg), and inferior colliculus (IC). The experiments were conducted with male Wistar rats of 270-290 g bw, catalepsy was evaluated by using the bar test. The injections into the PPTg and IC were without effect whereas the intra-SNc, intra-DS, and intra-LC administrations produced distinct cataleptic response. Thus, it was shown for the first time that the LC is a brain center capable of causing catalepsy. These data provide new insights into the neuroanatomical organization of the catalepsy-initiating mechanism and suggest the LC representing a potential target for therapeutic manipulations of extrapyramidal dysfunctions.

Ketamine-Induced Prefrontal Serotonin Release Is Mediated by Cholinergic Neurons in the Pedunculopontine Tegmental Nucleus.

Background: Ketamine rapidly elicits antidepressive effects in humans and mice in which serotonergic activity is involved. Although α4ß2 nicotinic acetylcholine receptor (α4ß2 nAChR) in the dorsal raphe nucleus plays a key role in the ketamine-induced prefrontal serotonin release, the source of cholinergic afferents, and its role is unclear. Methods: Prefrontal serotonin levels after ketamine injection were measured by microdialysis in rats. Electrolytic lesion of pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus was made with constant direct current. Results: Bilateral lesion of the pedunculopontine tegmental nucleus, but not laterodorsal tegmental nucleus, attenuated prefrontal serotonin release induced by systemic ketamine. Intra-pedunculopontine tegmental nucleus, but not intra-laterodorsal tegmental nucleus ketamine perfusion, increased prefrontal serotonin release. This increase was attenuated by intra-dorsal raphe nucleus injection of dihydro-ß-erythroidine, an α4ß2 nAChR antagonist, or NBQX, an AMPA receptor antagonist. Conclusions: These results suggest the ketamine-induced serotonin release in medial prefrontal cortex is mediated by cholinergic neurons projecting from pedunculopontine tegmental nucleus to dorsal raphe nucleus via α4ß2 nAChRs.

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.

REM sleep deprivation and dopaminergic D2 receptors modulation increase recognition memory in an animal model of Parkinson's disease.

Cognitive impairment is an important non-motor symptom of Parkinson's disease (PD). The neuronal death in nigrostriatal pathway is the main factor for motor symptoms and recent studies indicate a possible influence in non-motor symptoms as well. The pedunculopontine tegmental nucleus (PPT) and basal ganglia are closely related anatomically and functionally and, since they are affected by neurodegeneration in PD, they might be involved in recognition memory. To investigate this, we promoted an ibotenic acid lesion within the PPT or a rotenone lesion within substantia nigra pars compacta (SNpc) of Wistar rats, followed by 24h of REM sleep deprivation (REMSD). Then, we administered a dopaminergic D2 receptor agonist (piribedil, 3µg/µl), antagonist (raclopride, 10µg/µl) or vehicle (dimethylsulfoxide) directly in the striatum and the animals were submitted to the object recognition test (ORT). We observed that raclopride administration impaired object recognition memory as well as rotenone and ibotenic acid lesion. Interestingly, REMSD reversed the deleterious effects induced by these drugs. Also, raclopride administration after rotenone lesion allowed the animal to explore the new object for a longer time compared to the familiar object, suggesting that raclopride has a dual effect, dependent of the treatments. These findings suggest a role for PPT, SNpc and striatum in recognition memory and points the D2 receptors modulation and REMSD as possible targets for cognitive deficits in Parkinson's disease.

The pedunculopontine tegmentum controls renal sympathetic nerve activity and cardiorespiratory activities in nembutal-anesthetized rats.

Elevated renal sympathetic nerve activity (RSNA) accompanies a variety of complex disorders, including obstructive sleep apnea, heart failure, and chronic kidney disease. Understanding pathophysiologic renal mechanisms is important for determining why hypertension is both a common sequelae and a predisposing factor of these disorders. The role of the brainstem in regulating RSNA remains incompletely understood. The pedunculopontine tegmentum (PPT) is known for regulating behaviors including alertness, locomotion, and rapid eye movement sleep. Activation of PPT neurons in anesthetized rats was previously found to increase splanchnic sympathetic nerve activity and blood pressure, in addition to altering breathing. The present study is the first investigation of the PPT and its potential role in regulating RSNA. Microinjections of DL-homocysteic acid (DLH) were used to probe the PPT in 100-µm increments in Nembutal-anesthetized rats to identify effective sites, defined as locations where changes in RSNA could be evoked. A total of 239 DLH microinjections were made in 18 rats, which identified 20 effective sites (each confirmed by the ability to evoke a repeatable sympathoexcitatory response). Peak increases in RSNA occurred within 10-20 seconds of PPT activation, with RSNA increasing by 104.5 ± 68.4% (mean ± standard deviation) from baseline. Mean arterial pressure remained significantly elevated for 30 seconds, increasing from 101.6 ± 18.6 mmHg to 135.9 ± 36.4 mmHg. DLH microinjections also increased respiratory rate and minute ventilation. The effective sites were found throughout the rostal-caudal extent of the PPT with most located in the dorsal regions of the nucleus. The majority of PPT locations tested with DLH microinjections did not alter RSNA (179 sites), suggesting that the neurons that confer renal sympathoexcitatory functions comprise a small component of the PPT. The study also underscores the importance of further investigation to determine whether sympathoexcitatory PPT neurons contribute to adverse renal and cardiovascular consequences of diseases such as obstructive sleep apnea and heart failure.

Selective cholinergic depletion of pedunculopontine tegmental nucleus aggravates freezing of gait in parkinsonian rats.

Many patients of advanced Parkinson's disease (PD) suffer from intractable axial symptoms (severe gait and postural impairments), which were recently speculated to be more relevant to cholinergic degeneration in the brainstem than dopaminergic degeneration in the substantia nigra compacta (SNc). To investigate the role of the cholinergic cells of the pedunculopontine tegmental nucleus (PPTg) on motor deficits, especially the axial motor impairments, we measured and analyzed the gait performance of sham lesion rats, SNc dopaminergic lesion rats, PPTg cholinergic lesion rats, and combined lesion rats by using the CatWalk system. Motor performance of PPTg cholinergic lesion rats was also tested on the rotarod. Independent loss of cholinergic neurons in the PPTg did not induce gait disturbance in CatWalk, but PPTg lesion rats showed motor impairments on the rotarod when the demands of the motor task increased. Both SNc lesion rats and combined lesion rats displayed significant changes in many gait parameters, but the terminal dual stance increased much higher in combined lesion group than SNc lesion group. Furthermore, combined lesion rats showed more severe freezing of gait (FOG) than SNc lesion rats during behavioral re-evaluations after lesion. These results suggest that the PPTg cholinergic neurons play a vital role in the occurrence of FOG in PD.

Motor dysfunction and alterations in glutathione concentration, cholinesterase activity, and BDNF expression in substantia nigra pars compacta in rats with pedunculopontine lesion.

Pedunculopontine nucleus (PPN) has been considered a critically important region in the regulation of some of the physiological functions that fail during the progression of Parkinson's disease (PD). In this paper, the effects of unilateral neurotoxic lesion of the PPN [through the injection of N-methyl-d-aspartate (NMDA) solution (concentration: 0.1M; volume: 0.5µL)] in motor execution and gait disorders and the changes in cellular and molecular indicators in rat nigral tissue were evaluated. The motor execution was assessed using the beam test (BT) and the gait disorders by footprint test. Glutathione (GSH) concentrations, acetyl cholinesterase enzymatic activity (AChE EA), and brain-derived neurotrophic factor (BDNF) mRNA expression in nigral tissue were analyzed. NMDA-lesioned rats showed fine motor dysfunction with a significant increase in the slow (p≤0.01) and fast movement (p≤0.01) time and in path deviation (p≤0.01) on the smaller diameter beams. Moreover, NMDA-lesioned rats exhibited an imprecise path with moments of advances and setbacks, alternating with left and right deviations, suspensions, and inverted positions. Footprint test revealed slight gait disorders, which were manifested by a reduction in the left and right stride lengths, the intra-step distance, and the support area (p≤0.01). Biochemical studies showed that 48h after the PPN neurotoxic injury, the GSH concentrations and BDNF expression were significantly increased (p≤0.01). These variables returned to normal values 7days after the PPN lesion; the AChE EA showed a significant increase at this time. These functional changes in nigral tissue could be a plastic responses associated with early PD.

Direct presynaptic and indirect astrocyte-mediated mechanisms both contribute to endocannabinoid signaling in the pedunculopontine nucleus of mice.

The pedunculopontine nucleus (PPN), a cholinergic nucleus of the reticular activating system, is known to be involved in the regulation of sleep and wakefulness. Endogenous and exogenous cannabinoids, by systemic or local administration to the pedunculopontine nucleus, can both influence sleep. We previously demonstrated that activation of astrocytes by cannabinoid type 1 (CB1) receptor agonists was able to modulate the membrane potential of PPN neurons, even in the presence of blockers of fast synaptic neurotransmission. In the present work, we provide evidence that synaptic inputs of PPN neurons are also affected by activation of presynaptic and astrocytic CB1 receptors. Using slice electrophysiology combined with calcium imaging, optogenetics and immunohistochemistry, we revealed a direct presynaptic inhibitory action on inhibitory postsynaptic currents, along with a mild increase of excitatory postsynaptic currents during CB1 receptor stimulation. Besides inhibition of excitatory and inhibitory neurotransmission through stimulation of presynaptic CB1 receptors, astrocyte- and mGluR-dependent tonic inhibition and excitation also developed. The mild stimulatory action of CB1 receptor activation on excitatory neurotransmission is the combination of astrocyte-dependent tonic excitation on excitatory neurons and the canonical presynaptic CB1 receptor activation and consequential inhibition of excitatory synaptic neurotransmission, whereas the astrocyte-dependent stimulatory action was not observed in inhibitory neurotransmission within the PPN. Our findings demonstrate that endocannabinoids act in the PPN via a dual pathway, consisting of a direct presynaptic and an indirect, astrocyte-mediated component, regulating synaptic strength and neuronal activity via independent mechanisms.