GABAA receptors inhibit acetylcholine release in cat pontine reticular formation: implications for REM sleep regulation

This study used in vivo microdialysis in cat (n=12) to test the hypothesis that gamma aminobutyric acid A (GABAA) receptors in the pontine reticular formation (PRF) inhibit acetylcholine (ACh) release. Animals were anesthetized with halothane to hold arousal state constant. Six concentrations of the GABAA receptor antagonist bicuculline (0.03, 0.1, 0.3, 1, 3, and 10 mM) were delivered to a dialysis probe in the PRF, and endogenously released ACh was collected simultaneously. Bicuculline caused a concentration dependent increase in ACh release (maximal increase=345%; EC50=1.3 mM; r2=0.997). Co-administration of the GABAA receptor agonist muscimol prevented the bicuculline-induced increase in ACh release. In a second series of experiments, the effects of bicuculline (0.1, 0.3, 1, and 3 mM) on ACh release were examined without the use of general anesthesia. States of wakefulness, rapid-eye-movement (REM) sleep, and non-REM sleep were identified polygraphically before and during dialysis delivery of bicuculline. Higher concentrations of bicuculline (1 and 3 mM) significantly increased ACh release during wakefulness (36%), completely suppressed non-REM sleep, and increased ACh release during REM sleep (143%). The finding that ACh release in the PRF is modulated by GABAA receptors is consistent with the interpretation that inhibition of GABAergic transmission in the PRF contributes to the generation of REM sleep, in part, by increasing pontine ACh release.     

Microinjections of muscimol and bicuculline into the pontine reticular formation modify the sleep-waking cycle in the rat.

The role of gamma-aminobutyric acid (GABA) in the sleep-waking cycle was evaluated by means of microinjections of muscimol and bicuculline into the rat pontine reticular formation (PRF). Muscimol (20 ng) produced a marked increase in wakefulness (70%), a decrease in slow-wave sleep (SWS) (35%) and a remarkable delay in the onset of both SWS and paradoxical sleep, without modifying the percentage of the latter. Bicuculline (4 ng) shortened SWS latency by about 70%. These results suggest that GABAergic transmission in the PRF is involved in the regulation of sleep-waking cycle in the rat.     

M2 muscarinic receptor subtype in the feline medial pontine reticular formation modulates the amount of rapid eye movement sleep

Rapid eye movement (REM) sleep is generated, in part, by activating muscarinic cholinergic receptors (mAChRs) in the medial pontine reticular formation (mPRF). Molecular cloning has identified five mAChR subtypes, and this study tested the hypothesis that the M2 subtype in the mPRF modulates the amount of REM sleep. This hypothesis cannot be tested directly, due to lack of subtype selective muscarinic agonists. However, the amount of REM sleep can be enhanced by mPRF microinjection of a muscarinic agonist, and the relative potencies of muscarinic antagonists to block the REM sleep enhancement can be determined. Two muscarinic antagonists, methoctramine and 4-DAMP, were studied. Six concentrations of each antagonist were microinjected into the mPRF of conscious cat 15 min prior to the agonist bethanechol. Nonlinear regression analysis was used to calculate the dose of antagonist that caused a 50% inhibition (ID50) of bethanechol-induced REM sleep. Bethanechol significantly increased (442%) the amount of time spent in REM sleep. Both methoctramine and 4-DAMP significantly blocked the bethanechol-induced REM sleep increase, with an ID50 of 1.8 microM and 0.6 microM, respectively. The ID50 ratio for methoctramine-to-4-DAMP (3.0) was similar to the affinity ratio of methoctramine-to-4-DAMP only at the M2 subtype (3.5), suggesting that the M2 subtype in the mPRF modulates the amount of REM sleep. This study also tested the null hypothesis that sleep-dependent respiratory depression evoked by mPRF cholinomimetics would not be antagonized by pretreatment of the mPRF with muscarinic antagonists. Neither methoctramine nor 4-DAMP antagonized the bethanechol-induced decrease in respiratory rate.     

Infusion of adenylyl cyclase inhibitor SQ22,536 into the medial pontine reticular formation of rats enhances rapid eye movement sleep

Microinjection of cholinergic and adenosinergic agonists into the medial pontine reticular formation of rats produces long lasting increases in the time spent in rapid eye movement sleep. Several G-protein-coupled muscarinic and adenosinergic receptors share a common action of inhibition of adenylyl cyclase and inhibition of cyclic adenosine monophosphate. Inhibition of cyclic adenosine monophosphate has been implicated in the mechanism of rapid eye movement sleep induction in the cat. We sought to determine whether a direct inhibitor of adenylyl cyclase microinjected into the rat reticular formation at sites where muscarinic and adenosinergic agonists are effective in producing long lasting elevations in rapid eye movement sleep also result in similar effects on the sleep/wake cycle. The caudal, oral pontine reticular formation was unilaterally infused with 60 nl volumes of carbachol (0.1-1.1mM) and N(6)-cyclohexyladenosine (0.1mM) each within 1h of lights on. Sites effective for significantly elevating rapid eye movement sleep for the 8h following microinjection of both receptor agonists were additionally injected with the adenylyl cyclase inhibitor, SQ22,536 (0.1M). Pontine injections of SQ22,536 resulted in significant mean increases in rapid eye movement sleep time and rapid eye movement sleep period frequency at all of these sites. As with the receptor agonists, SQ22,536 did not alter latency to rapid eye movement sleep onset. Rapid eye movement sleep amounts were observed to be significantly elevated by SQ22,536 at two days, but not at four days, following a single microinjection.These data implicate inhibition of cyclic adenosine monophosphate in the pons of the rat as a mechanism involved in the long-term modulation of rapid eye movement sleep. This mechanism may underlie the homeostatic regulation exhibited by this sleep-state.     

Enhancement of dorsal raphe discharge by medial pontine reticular formation stimulation depends on behavioral state

Single cell activity was recorded with microwire electrodes from the dorsal raphe nucleus (DRN) of cats across multiple cycles of sleep and wakefulness. Electrical stimulation of the medial pontine reticular formation (mPRF) produced a 10-fold enhancement of DRN discharge during wakefulness and slow wave sleep but not during desynchronized sleep. The neuronal mechanisms likely to mediate the state-dependent effects of mPRF stimulation are discussed with regard to the hypothesis that DRN plays a major role in behavioral state control.     

Postnatal development of spontaneous neuronal discharges in the pontine reticular formation of free-moving rats during sleep and wakefulness

Summary A developmental study has been made of spontaneous neuronal activity within the pontine reticular formation (giant cell field: FTG) of the rat between one week and one month after birth. Through day 14, the recorded FTG neurons discharged more frequently during quiet sleep (QS) than was generally true in older animals. In addition, they were active to the same extent during active-sleep (AS) as during waking-with-movements (AW). In contrast, most of the cells recorded from day 15 on were considerably more active during AS and AW, relative to the QS level, than had hitherto been the case. This new class of neurons, in turn, fell into two sub-groups, one of which was most active during AW while the other was more active during AS. Clomipramine selectively suppressed AS along with the neuronal activity patterns associated with it, and in many cases the QS firing level was even lower than it had been prior to the injection. It is concluded that FTG unit activity is an excellent monitor for controlling the effectiveness of experimental manipulations of AS but is probably not involved in its generation.     

REM sleep without atonia after lesions of the medial medulla

Rapid eye movement (REM) sleep is normally accompanied by a complete suppression of tone in the antigravity musculature. Pontine lesions have been shown to block this suppression, producing a syndrome of REM sleep without atonia. We now report that glutamate-induced lesions of the medial medulla, including the nucleus magnocellularis, caudal nucleus gigantocellularis and rostral nucleus paramedianus, produce REM sleep without atonia. These nuclei may function as part of a ponto-medullary system suppressing muscle tone in REM sleep.     

Ketamine and MK-801 decrease acetylcholine release in the pontine reticular formation,slow breathing,and disrupt sleep

STUDY OBJECTIVES: Ketamine induces a dissociated state of consciousness by binding to the phencyclidine binding site within the ion channel gated by the N-methyl-D-aspartate (NMDA) receptor. The brain regions and neurotransmitter systems mediating ketamine-induced alterations in arousal remain incompletely understood. This study used in vivo microdialysis to test the hypothesis that ketamine alters acetylcholine (ACh) release in the medial pontine reticular formation (mPRF). DESIGN: Acetylcholine (ACh) release, sleep, and breathing were quantified following systemic ketamine administration. Microdialysis was used to deliver the NMDA-channel blocker dizocilpine maleate (MK-801) and the R(-)-isomer of ketamine into the mPRF while measuring ACh release. SETTING: N/A PARTICIPANTS: N/A INTERVENTIONS: N/A MEASUREMENTS AND RESULTS: Systemically administered ketamine disrupted normal sleep-cycle organization, reduced mPRF ACh release, and significantly slowed rate of breathing. Dialysis delivery of MK-801 to the mPRF significantly decreased respiratory rate and mPRF ACh release. Dialysis delivery to the mPRF of the R(-)-ketamine isomer significantly decreased mPRF ACh release. CONCLUSIONS: Decreased mPRF ACh release caused by systemically administered ketamine was mimicked by mPRF dialysis delivery of MK-801 and the R(-)-ketamine isomer. These data are consistent with the conclusion that systemically administered ketamine may alter arousal and breathing, in part, by altering cholinergic neurotransmission in the mPRF.     

Hypocretin and GABA interact in the pontine reticular formation to increase wakefulness

Study Objectives:

Hypocretin-1/orexin A administered directly into the oral part of rat pontine reticular formation (PnO) causes an increase in wakefulness and extracellular γ-aminobutyric acid (GABA) levels. The receptors in the PnO that mediate these effects have not been identified. Therefore, this study tested the hypothesis that the increase in wakefulness caused by administration of hypocretin-1 into the PnO occurs via activation of GABA_A receptors and hypocretin receptors.

Design:

Within/between subjects.

Setting:

University of Michigan.

Patients or Participants:

Twenty-three adult male Crl:CD*(SD) (Sprague Dawley) rats.

Interventions:

Microinjection of hypocretin-1, bicuculline (GABA_A receptor antagonist), SB-334867 (hypocretin receptor-1 antagonist), and Ringer solution (vehicle control) into the PnO.

Measurements and Results:

Hypocretin-1 caused a significant concentration-dependent increase in wakefulness and decrease in rapid eye movement (REM) sleep and non-REM (NREM) sleep. Coadministration of SB-334867 and hypocretin-1 blocked the hypocretin-1–induced increase in wakefulness and decrease in both the NREM and REM phases of sleep. Coadministration of bicuculline and hypocretin-1 blocked the hypocretin-1–induced increase in wakefulness and decrease in NREM sleep caused by hypocretin-1.

Conclusion:

The increase in wakefulness caused by administering hypocretin-1 to the PnO is mediated by hypocretin receptors and GABA_A receptors in the PnO. These results show for the first time that hypocretinergic and GABAergic transmission in the PnO can interact to promote wakefulness.

Citation:

Brevig HN; Watson CJ; Lydic R; Baghdoyan HA. Hypocretin and GABA interact in the pontine reticular formation to increase wakefulness. SLEEP 2010;33(10):1285-1293.     

Serotonin1 and serotonin2 receptors hyperpolarize and depolarize separate populations of medial pontine reticular formation neurons in vitro.

The action of serotonin on medial pontine reticular formation neurons was examined using intracellular electrophysiological methods in rat brainstem slices in vitro. A hyperpolarization associated with a decrease in input resistance was elicited by serotonin in 34% of the neurons, and a depolarization associated with an increase in input resistance was produced in 56% of the neurons. Both responses persisted in the presence of tetrodotoxin. The hyperpolarization resulted from a steady-state increase in outward current which varied with the external potassium concentration in a manner consistent with a conductance increase primarily to this ion. This response was mimicked by the serotonin1 agonist, 5-carboxamidotryptamine, as well as by the serotonin1a agonist, 8-hydroxy-dipropyl aminotetralin hydrobromide, and was blocked by spiperone, an antagonist of serotonin1 sites. The depolarization resulted from a steady-state decrease in outward current which varied with external potassium. The depolarization was mimicked by the serotonin2 agonist, alpha-methyl-5-hydroxytryptamine, and was blocked by the serotonin2 antagonist, ketanserin. Neither of these agents had any effect upon serotonin-induced hyperpolarizations. In conclusion, the excitability of medial pontine reticular formation neurons is influenced by serotonin acting to increase or decrease potassium conductance(s). These opposing effects reflect actions on distinct serotonin receptor subtypes that are segregated to distinct populations of medial pontine reticular formation neurons.     

A quantitative study of the brainstem cholinergic projections to the ventral part of the oral pontine reticular nucleus (REM sleep induction site) in the cat

The ventral part of the cat oral pontine reticular nucleus (vRPO) is the site in which microinjections of small dose and volume of cholinergic agonists produce long-lasting rapid eye movement sleep with short latency. The present study determined the precise location and proportions of the cholinergic brainstem neuronal population that projects to the vRPO using a double-labeling method that combines the neuronal tracer horseradish peroxidase–wheat germ agglutinin with choline acetyltransferase immunocytochemistry in cats. Our results show that 88.9% of the double-labeled neurons in the brainstem were located, noticeably bilaterally, in the cholinergic structures of the pontine tegmentum. These neurons occupied not only the pedunculopontine and laterodorsal tegmental nuclei, which have been described to project to other pontine tegmentum structures, but also the locus ceruleus complex principally the locus ceruleus and peri-, and the parabrachial nuclei. Most double-labeled neurons were found in the pedunculopontine tegmental nucleus and locus ceruleus complex and, much less abundantly, in the laterodorsal tegmental nucleus and the parabrachial nuclei. The proportions of these neurons among all choline acetyltransferase positive neurons within each structure were highest in the locus ceruleus complex, followed in descending order by the pedunculopontine and laterodorsal tegmental nuclei and then, the parabrachial nuclei. The remaining 11.1% of double-labeled neurons were found bilaterally in other cholinergic brainstem structures: around the oculomotor, facial and masticatory nuclei, the caudal pontine tegmentum and the praepositus hypoglossi nucleus. The disperse origins of the cholinergic neurons projecting to the vRPO, in addition to the abundant noncholinergic afferents to this nucleus may indicate that cholinergic stimulation is not the only or even the most decisive event in the generation of REM sleep.     

Transplantation of hypocretin neurons into the pontine reticular formation: preliminary results

STUDY OBJECTIVES: The sleep disorder narcolepsy is now considered a neurodegenerative disease because there is a massive loss of neurons containing the neuropeptide, hypocretin, and because narcoleptic patients have very low cerebrospinal fluid levels of hypocretin. Transplants of various cell types have been used to induce recovery in a variety of neurodegenerative animal models. In models such as Parkinson disease, cell survival has been shown to be small but satisfactory. Currently, there are no data indicating whether hypocretin neurons can survive when grafted into host tissue. Here we examined the survival of hypocretin-containing neurons grafted into the pontine reticular formation, a region traditionally regarded to be key for rapid eye movement sleep generation. DESIGN: In 2 experiments, a suspension of cells from the posterior hypothalamus of 8- to 10-day old rat pups was injected into the pons (midline, at the level of the locus coeruleus) of adult rats. Control rats received cells from the cerebellum, tissue that is devoid of hypocretin neurons. In the first experiment (n = 33), the adult rats were sacrificed 1, 3, 6, 12, 24, or 36 days after transplant, and cryostat-cut coronal sections of the brainstem were examined for presence of hypocretin-immunoreactive neurons. In the second experiment (n = 9), the transplant medium was modified to include agents that stimulate cell growth, and recipient rats were sacrificed 9, 12, and 36 days after receiving the graft. SETTINGS: Basic neuroscience research laboratory. MEASUREMENTS AND RESULTS: In the first experiment, clearly defined hypocretin-immunoreactive containing somata and varicosities were visible in pons of rats sacrificed 1 day after grafting of posterior hypothalamic cells but not in rats receiving cerebellum tissue. The hypocretin-immunoreactive somata were not visible in rats sacrificed at 12, 24, or 36 days, indicating that the neurons had died. However, in the second experiment, where enriched transplant medium was used, clearly defined hypocretin-immunoreactive somata with processes and varicosities were present in the graft zone 36 days after implant. These somata were similar in size and appearance to adult rat hypocretin-immunoreactive neurons. CONCLUSIONS: These results indicate that hypocretin neurons obtained from rat pups can be grafted into a host brain, and efforts should be made to increase survival of these neurons.     

Catalepsy induced by carbachol microinjected into the pontine reticular formation of rats

Rats were implanted stereotaxically with permanent guide tubes aimed at the pontine reticular formation. Carbachol was microinjected in the awake, freely moving rats. Catalepsy was evaluated with the horizontal bar test. Intense and long-lasting, dose-dependent catalepsy was observed following microinjections of 2-8 micrograms of carbachol. Pretreatment with intraperitoneally administered scopolamine significantly reduced the cataleptogenic effect of carbachol. These results show that the pontine reticular formation is part of the neuroanatomical substrate of catalepsy induced by cholinergic drugs.     

Effects of pontine reticular formation lesions on optokinetic head nystagmus in rats

Summary Electrolytic lesions were produced in the pontine reticular formation (PRF) of adult hooded rats. Unilateral lesions abolished quick phases of optokinetic head nystagmus to the side of the lesion. Some lesions also had temporary effects on slow phases of optokinetic head nystagmus. Effects of bilateral lesions were similar, except that they affected head movements in both directions. A class of fast head movements abolished by PRF lesions thus emerges that is analogous to the class of rapid eye movements abolished by similar lesions in other species, a finding that can be related to the coupling which has been observed between fast head and eye movements.Supported by NIH grant HEW PHS 2 R01 NS11671 to P.T. and NSF grant SER 76-18255This study formed part of the doctoral dissertation of D.W.S.     

Properties and interconnections of trigeminal interneurons of the lateral pontine reticular formation in the rat.

Numerous evidence suggests that interneurons located in the lateral tegmentum at the level of the trigeminal motor nucleus contribute importantly to the circuitry involved in mastication. However, the question of whether these neurons participate actively to genesis of the rhythmic motor pattern or simply relay it to trigeminal motoneurons remains open. To answer this question, intracellular recordings were performed in an in vitro slice preparation comprising interneurons of the peritrigeminal area (PeriV) surrounding the trigeminal motor nucleus (NVmt) and the parvocellular reticular formation ventral and caudal to it (PCRt). Intracellular and extracellular injections of anterograde tracers were also used to examine the local connections established by these neurons. In 97% of recordings, electrical stimulation of adjacent areas evoked a postsynaptic potential (PSP). These PSPs were primarily excitatory, but inhibitory and biphasic responses were also induced. Most occurred at latencies longer than those required for monosynaptic transmission and were considered to involve oligosynaptic pathways. Both the anatomical and physiological findings show that all divisions of PeriV and PCRt are extensively interconnected. Most responses followed high-frequency stimulation (50 Hz) and showed little variability in latency indicating that the network reliably distributes inputs across all areas. In all neurons but one, excitatory postsynaptic potentials (EPSPs) or inhibitory postsynaptic potentials (IPSPs) were also elicited by stimulation of NVmt, suggesting the existence of excitatory and inhibitory interneurons within the motor nucleus. In a number of cases, these PSPs were reproduced by local injection of glutamate in lieu of the electrical stimulation. All EPSPs induced by stimulation of PeriV, PCRt, or NVmt were sensitive to ionotropic glutamate receptor antagonists 6-cyano-7-dinitroquinoxaline and D,L-2-amino-5-phosphonovaleric acid, while IPSPs were blocked by bicuculline and strychnine, antagonists of GABA(A) and glycine receptors. Examination of PeriV and PCRt intrinsic properties indicate that they form a fairly uniform network. Three types of neurons were identified on the basis of their firing adaptation properties. These types were not associated with particular regions. Only 5% of all neurons showed bursting behavior. Our results do not support the hypothesis that neurons of PeriV and PCRt participate actively to rhythm generation, but suggest instead that they are driven by rhythmical synaptic inputs. The organization of the network allows for rapid distribution of this rhythmic input across premotoneuron groups.