Nucleus Pontis Oralis Research Articles

Progressive generalized tonic-clonic seizures in a transgenic mouse model of adult-onset epilepsy: Implications for morphological changes in cortico-limbic and brainstem structures

Video/cortical electroencephalography (EEG) is monitored to assess progressive severity of generalized tonic clonic seizures (GTCSs) in a transgenic mouse model of adult-onset epilepsy with increased death risk. The mice overexpress the brain derived neurotrophic factor (BDNF) in the forebrain under the calcium/calmodulin dependent protein kinase 2a (termed TgBDNF) and develop GTCSs in response to tail suspension/cage agitation stimulation at 3–4 months of age. With successive GTCSs (a total of 16 across 10 weeks of assessment), seizures became more severe as evidenced by increased duration of postictal generalized EEG suppression (PGES) associated with loss of posture/consciousness. Mice also developed spike wave discharges with behavioral arrest during the seizure recovery that increased in duration as a function of number of GTCSs. Overall seizure duration (from preictal spike to offset of PGES) and ictal spectral power (full spectra) were also increased. Half of the TgBDNF mice expired following a long period of PGES at the last recorded GTCS. Seizure-evoked general arousal impairment was associated with a striking decrease in total number of gigantocellular neurons of the brainstem nucleus pontis oralis along with increase in volumes of the anterior cingulate cortex and dorsal dentate gyrus in severely convulsive TgBDNF mice compared to litter-matched WT controls and non-convulsive TgBDNF mice. The latter effect was accompanied with an increase in total number of hippocampal granule neurons. These results provide structure-function associations in an animal model of adult-onset GTCSs that progressively increase in severity with clinical relevance for sudden unexpected death following generalized seizures.

Arousal system stimulation and anesthetic state alter visuoparietal connectivity.

Cortical information processing is under the precise control of the ascending arousal system (AAS). Anesthesia suppresses cortical arousal that can be mitigated by exogenous stimulation of the AAS. The question remains to what extent cortical information processing is regained by AAS stimulation. We investigate the effect of electrical stimulation of the nucleus Pontis Oralis (PnO), a distinct source of ascending AAS projections, on cortical functional connectivity (FC) and information storage at mild, moderate, and deep anesthesia. Local field potentials (LFPs) recorded previously in the secondary visual cortex (V2) and the adjacent parietal association cortex (PtA) in chronically instrumented unrestrained rats. We hypothesized that PnO stimulation would induce electrocortical arousal accompanied by enhanced FC and active information storage (AIS) implying improved information processing. In fact, stimulation reduced FC in slow oscillations (0.3-2.5 Hz) at low anesthetic level and increased FC at high anesthetic level. These effects were augmented following stimulation suggesting stimulus-induced plasticity. The observed opposite stimulation-anesthetic impact was less clear in the γ-band activity (30-70 Hz). In addition, FC in slow oscillations was more sensitive to stimulation and anesthetic level than FC in γ-band activity which exhibited a rather constant spatial FC structure that was symmetric between specific, topographically related sites in V2 and PtA. Invariant networks were defined as a set of strongly connected electrode channels, which were invariant to experimental conditions. In invariant networks, stimulation decreased AIS and increasing anesthetic level increased AIS. Conversely, in non-invariant (complement) networks, stimulation did not affect AIS at low anesthetic level but increased it at high anesthetic level. The results suggest that arousal stimulation alters cortical FC and information storage as a function of anesthetic level with a prolonged effect beyond the duration of stimulation. The findings help better understand how the arousal system may influence information processing in cortical networks at different levels of anesthesia.

TREM2 Deficiency Disrupts Network Oscillations Leading to Epileptic Activity and Aggravates Amyloid-β-Related Hippocampal Pathophysiology in Mice.

Genetic mutations in triggering receptor expressed on myeloid cells-2 (TREM2) have been strongly associated with increased risk of developing Alzheimer's disease (AD) and other progressive dementias. In the brain, TREM2 protein is specifically expressed on microglia suggesting their active involvement in driving disease pathology. Using various transgenic AD models to interfere with microglial function through TREM2, several recent studies provided important data indicating a causal link between TREM2 and underlying amyloid-β (Aβ) and tau pathology. However, mechanisms by which TREM2 contributes to increased predisposition to clinical AD and influences its progression still remain largely unknown. Our aim was to elucidate the potential contribution of TREM2 on specific oscillatory dynamic changes associated with AD pathophysiology. Spontaneous and brainstem nucleus pontis oralis stimulation-induced hippocampal oscillation paradigm was used to investigate the impact of TREM2 haploinsufficiency TREM2(Het) or total deficiency TREM2(Hom) on hippocampal network function in wild-type and Aβ overproducing Tg2576 mice under urethane anesthesia. Partial (TREM2(Het)) or total (TREM2(Hom)) deletion of TREM2 led to increased incidence of spontaneous epileptiform seizures in both wild-type and Tg2576 mice. Importantly, deficiency of TREM2 in Tg2576 mice significantly diminished power of theta oscillation in the hippocampus elicited by brainstem-stimulation compared to wild-type mice. However, it did not affect hippocampal theta-phase gamma-amplitude coupling significantly, since over a 60%reduction was found in coupling in Tg2576 mice regardless of TREM2 function. Our findings indicate a role for TREM2-dependent microglial function in the hippocampal neuronal excitability in both wild type and Aβ overproducing mice, whereas deficiency in TREM2 function exacerbates disruptive effects of Aβ on hippocampal network oscillations.

027 GABAergic Neurons in the Dorsal Raphe Nucleus Are under the Influence of GABAergic Inputs from the Nucleus Pontis Oralis

Abstract Introduction Our previous study has shown that there is a direct connection between GABAergic neurons in the nucleus pontis oralis (NPO) and neurons of the dorsal raphe nucleus (DR), providing a morphological basis for the hypothesis that GABAergic inhibitory processes in NPO play an important role in the generation and maintenance of wakefulness as well as active (REM) sleep through the interaction with neurons in the DR. However, the target of such a GABAergic projection from the NPO within the DR is unknown. In the present study, a double-fluorescent labeling technique was employed to examine the target of GABAergic inputs to the DR. Methods Adult cats were deeply anesthetized and perfused transcardially. Subsequently, the brainstem containing the DR was removed, postfixed and cut into 15 μm coronal sections with a Reichert-Jung cryostat. The sections were immunostained with antibodies against GABA-A or GABA-B receptors and GABA following the procedure of double fluorescence immunohistochemistry. Results Under fluorescence microscopy, a large number of neurons were labeled with antibodies against either GABA-A receptor or GABA-B receptor. In addition, neurons labeled with antibody against GABA were observed in the DR. With double fluorescence immunohistochemical techniques, some neurons labeled by anti-GABA antibody were also stained with antibodies against GABA-A or GABA-B receptors. Conclusion The expression of GABA-A or GABA-B receptors by GABAergic neurons in the DR indicates that GABAergic neurons in the DR receive GABAergic inputs. Our previous study has demonstrated that these GABAergic inputs are from the NPO. These data provide a morphological foundation to support our hypothesis that, during wakefulness, NPO GABAergic “Executive” neurons suppress “Second-Order” GABAergic neurons in the DR, which, in turn, activate (disinhibit) serotonergic wake-on neurons in this nucleus. Support (if any) NS092383

Neostigmine Microinjected into Prefrontal Cortex of C57BL/6J (B6) Mice Stimulates Breathing

The prefrontal cortex modulates breathing (Neuroscience 123: 17, 2004; J Physiol 591: 6069, 2013) via neurochemical mechanisms that remain to be fully characterized. This study is testing the hypothesis that microinjection of neostigmine bromide, an acetylcholinesterase inhibitor, into the prefrontal cortex of freely behaving mice alters breathing. All procedures involving mice were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. Adult male mice (n = 8) were anesthetized and implanted with a 26-gauge guide cannula aimed at the medial prefrontal cortex. One week after recovering from surgery, mice were microinjected (50 nL) with saline (vehicle control) and two concentrations of neostigmine (0.005 and 0.016 nmol/50 nL; 1.5 and 4.8 ng/50 nL). Each mouse received saline and neostigmine in a repeated measures within-subjects design. Respiratory rate (RR) expressed as breaths/min was quantified using whole-body plethysmography. Repeated measures one-way ANOVA showed neostigmine significantly (F(1.9,13.9) = 6.3, p = 0.0116) altered RR. Relative to saline, RR was increased 11.9% by 0.005 nmol and 18.2% by 0.016 nmol neostigmine. Dunnett's post hoc comparisons test revealed that 0.016 nmol significantly (p < 0.05) increased RRwhen compared to saline. Cohen's d = 1.34 for saline vs. 0.016 nmol neostigmine. Histological analyses of microinjection sites confirmed drug delivery into the prelimbic region of the prefrontal cortex. Mean ± S.D. coordinates of the injection sites were 1.8 ± 0.4 mm anterior, 0.3 ± 0.1 mm lateral, and 2.6 ± 0.6 mm ventral relative to bregma (Paxinos and Franklin, 2008). The mechanism by which cholinergic transmission in the prefrontal cortex stimulates breathing remains to be identified. We speculate that the neostigmine-induced increase in RR evoked from the prefrontal cortex may be modulated by projections from the prefrontal cortex to arousal promoting regions of the nucleus pontis oralis (PnO) (J. Neurosci. 29: 871, 2009). Neuroanatomical studies show that the PnO shares reciprocal connections with respiratory neurons in the ponto-medullary brainstem (Lung Cell. Mol. Physiol. 2: L251, 1995). These anatomical data are consistent with functional studies showing that respiratory rate in B6 mice significantly increased in tandem with increases in prefrontal cortex ACh release (Neuroscience 123: 17, 2004). Considered together, the present results support the interpretation that cholinergic neurotransmission in the prefrontal cortex stimulates the drive to breathe. Ongoing studies are further characterizing the site-dependent and concentration-dependent stimulation of breathing by cholinergic transmission in the prefrontal cortex.

Nicotinic Receptors in the Brainstem Ascending Arousal System in SIDS With Analysis of Pre-natal Exposures to Maternal Smoking and Alcohol in High-Risk Populations of the Safe Passage Study.

Pre-natal exposures to nicotine and alcohol are known risk factors for sudden infant death syndrome (SIDS), the leading cause of post-neonatal infant mortality. Here, we present data on nicotinic receptor binding, as determined by 125I-epibatidine receptor autoradiography, in the brainstems of infants dying of SIDS and of other known causes of death collected from the Safe Passage Study, a prospective, multicenter study with clinical sites in Cape Town, South Africa and 5 United States sites, including 2 American Indian Reservations. We examined 15 pons and medulla regions related to cardiovascular control and arousal in infants dying of SIDS (n = 12) and infants dying from known causes (n = 20, 10 pre-discharge from time of birth, 10 post-discharge). Overall, there was a developmental decrease in 125I-epibatidine binding with increasing postconceptional age in 5 medullary sites [raphe obscurus, gigantocellularis, paragigantocellularis, centralis, and dorsal accessory olive (p = 0.0002–0.03)], three of which are nuclei containing serotonin cells. Comparing SIDS with post-discharge known cause of death (post-KCOD) controls, we found significant decreased binding in SIDS in the nucleus pontis oralis (p = 0.02), a critical component of the cholinergic ascending arousal system of the rostral pons (post-KCOD, 12.1 ± 0.9 fmol/mg and SIDS, 9.1 ± 0.78 fmol/mg). In addition, we found an effect of maternal smoking in SIDS (n = 11) combined with post-KCOD controls (n = 8) on the raphe obscurus (p = 0.01), gigantocellularis (p = 0.02), and the paragigantocellularis (p = 0.002), three medullary sites found in this study to have decreased binding with age and found in previous studies to have abnormal indices of serotonin neurotransmission in SIDS infants. At these sites, 125I-epibatidine binding increased with increasing cigarettes per week. We found no effect of maternal drinking on 125I-epibatidine binding at any site measured. Taken together, these data support changes in nicotinic receptor binding related to development, cause of death, and exposure to maternal cigarette smoking. These data present new evidence in a prospective study supporting the roles of developmental factors, as well as adverse exposure on nicotinic receptors, in serotonergic nuclei of the rostral medulla—a finding that highlights the interwoven and complex relationship between acetylcholine (via nicotinic receptors) and serotonergic neurotransmission in the medulla.

0067 An Anatomic Substrate for GABAergic Processes to Suppress Active Sleep and Promote Wakefulness in the Nucleus Pontis Oralis

Abstract Introduction Our previous electrophysiologic data have provided compelling evidence that GABAergic processes in the nucleus pontis oralis (NPO) play a critical role in the generation and maintenance of wakefulness as well as active (REM) sleep (AS). We therefore hypothesized that one of the neuronal mechanisms of GABA actions in the NPO to promote wakefulness and suppress AS is due to a direct GABAergic inhibition of NPO neurons that generate AS (AS-generator neurons). However, the anatomical substrate for this inhibition is undetermined. Accordingly, the present study was undertaken to examine whether there is any direct interaction between GABAergic neurons and glutamatergic AS-generator neurons in the NPO. Methods Adult cats were deeply anesthetized and perfused transcardially. The brainstem containing the NPO was removed, postfixed and cut into 15 μm coronal sections with a Reichert-Jung cryostat. The sections were incubated with a mixture of a rabbit polyclonal antibodies against glutamine and GABA following the procedure of double fluorescence immunohistochemistry. Results There was a large number of neuronal somata labeled by anti-glutamine antibody and terminals labeled by anti-GABA antibody in the NPO. These glutamine-positive neurons were medium to large, multipolar cells (&amp;gt; 20 μm), which resemble glutamatergic, AS-generator neurons that have been previously identified in the NPO. Specifically, majority of glutamatergic neuronal somata were closely apposed by multiple GABAergic terminals, indicating that AS-generator neurons in the NPO receive direct GABAergic inputs. Conclusion The present results demonstrate that a direct connection exists between glutamatergic AS-generator neurons and GABAergic processes in the NPO. These data provide the anatomical evidence which supports our hypothesis that the pontine GABAergic control of wakefulness and active sleep is partially mediated via GABAergic processes project to NPO AS-generator neurons that suppress the activity of these cells. Support NS092383

Neurophysiological signals as predictive translational biomarkers for Alzheimer\u2019s disease treatment: effects of donepezil on neuronal network oscillations in TgF344-AD rats

BackgroundTranslational research in Alzheimer’s disease (AD) pathology provides evidence that accumulation of amyloid-β and hyperphosphorylated tau, neuropathological hallmarks of AD, is associated with complex disturbances in synaptic and neuronal function leading to oscillatory abnormalities in the neuronal networks that support memory and cognition. Accordingly, our recent study on transgenic TgF344-AD rats modeling AD showed an age-dependent reduction of stimulation-induced oscillations in the hippocampus, and disrupted long-range connectivity together with enhanced neuronal excitability in the cortex, reflected in greatly increased expression of high-voltage spindles, an epileptic absence seizure-like activity. To better understand the translational value of observed oscillatory abnormalities in these rats, we examine here the effects of donepezil, an acetylcholine esterase inhibitor clinically approved for AD treatment.MethodsBrainstem nucleus pontis oralis stimulation-induced hippocampal oscillations were recorded under urethane anesthesia in adult (6-month-old) and aged (12-month-old) TgF344-AD and wild-type rats. Spontaneous cortical activity was monitored in a cohort of freely behaving aged rats implanted with frontal and occipital cortical electroencephalography (EEG) electrodes.ResultsSubcutaneous administration of donepezil significantly augmented stimulation-induced hippocampal theta oscillation in aged wild-type rats and both adult and aged TgF344-AD rats, which have been previously shown to have diminished response to nucleus pontis oralis stimulation. Moreover, in adult TgF344-AD rats, donepezil also significantly increased theta phase-gamma amplitude coupling in the hippocampus during stimulation. However, neither of these effects were significantly changed in adult wild-type rats. Under freely behaving conditions, donepezil treatment had the opposite effect on cortical oscillatory connectivity in TgF344-AD and wild-type rats, and it reduced the occurrence of high-voltage spindle activity in TgF344-AD rats.ConclusionsTogether, these results imply that pharmacologically enhancing cholinergic tone with donepezil could partially reverse oscillatory abnormalities in TgF344-AD rats, which is in line with its clinical effectiveness in AD patients. Therefore, our study suggests good translational opportunities for these neurophysiological signals recorded in TgF344-AD rats, and their application could be considered in drug discovery efforts for developing therapies with disease-modifying potential.

0070 Anatomical Evidence of Direct Hypocretinergic Control of GABAergic Neurons in the Nucleus Pontis Oralis

A large number of studies have implicated the hypocretin (orexin) system as a critical regulator of sleep/wake states as well as feeding behavior and reward processes. On the other hand, a plethora of data demonstrates that GABAergic inhibitory processes in the nucleus pontis oralis (NPO) play an important role in the generation and maintenance of wakefulness as well as active (REM) sleep. Accordingly, the present study was undertaken to investigate whether there is any direct interaction of the hypocretinergic neurons and GABAergic neurons that comprise the pontine GABAergic system which functions to promote wakefulness and suppress active sleep in the NPO. Adult cats were deeply anesthetized and perfused transcardially. The brainstem containing the NPO was removed, postfixed and cut into 15 µm coronal sections with a Reichert-Jung cryostat. The sections were immunostained with antibodies against hypocretin receptor 1 (Hcrtr1) or 2 (Hcrtr2) and GABA following the procedure of double fluorescence immunohistochemistry. Under fluorescence microscopy, neurons exhibiting both GABA and Hcrtr1 or GABA and Hcrtr2 immunoreactivities were observed in the NPO, indicating that GABAergic neurons in the NPO receive direct hypocretinergic inputs via both receptors. However, the number of Hcrtr1 immunoreactivities that expressed on NPO GABAergic neurons was greater than Hcrtr2. The present results demonstrate that GABAergic neurons in the NPO is under direct influence of the hypocretinergic inputs and such a direct control is mediated by both hypocretin receptors. Therefore, we suggest that the hypocretinergic control of wakefulness and active sleep is partly mediated via direct activation of pontine GABAergic neurons by the hypocretins. NS092383.

CORRELATION OF ACETYLCHOLINE LEVELS WITH NEURONAL OSCILLATIONS IN THE BRAIN: A PRECLINICAL COMPARISON STUDY

Acetylcholine is commonly monitored in brain regions as a neurochemical evidence for putative cognitive enhancers. Similarly, theta and gamma oscillations are assessed as a pharmacodynamic biomarkers associated with cognition. However, comparison of these two models is not thoroughly evaluated in the literature. Correlation observed in these two cognitive biomarkers will be helpful in screening of compounds in early drug discovery. Brain microdialysis was carried out in hippocampus of freely moving rats. Microdialysis probe with 4 mm dialysis membrane was implanted steriotaxically and dialysis experiments began after 4–5 days of surgical recovery. Dialysates were subjected for quantification of acetylcholine using LC-MS/MS. Theta modulation studies were conducted in anesthetized rats. Electrical stimulations were applied to the nucleus pontis oralis and power of oscillatory activity was measured in hippocampus CA1 region. Modulation in oscillatory gamma activity was measured using multi-channel telemetric device (Data Sciences International). Acetylcholinesterase inhibitors like donepezil and rivastigmine; muscarinic receptor selective positive allosteric modulators were used as a tool compounds. Donepezil produced dose dependent increase in acetylcholine levels with mean maximum increase of 700% at the highest tested dose. However, rivastigmine produced increase in acetylcholine levels lesser than donepezil. M1-PAM, although produced moderate increase in the neurotransmitter levels, combination with donepezil had a beneficial effects on the acetylcholine increase. Increase in the low frequency, theta oscillations and high frequency, gamma oscillations were observed only when the treatment produced more than 500% increase in acetylcholine levels. Results from this multi-model comparison produced a good correlation between the acetylcholine increase and the neuronal oscillations. However, neurotransmitter modulation is more sensitive indicator of pharmacodynamic effect than the changes observed with NPO stimulation model.

0138 DIFFERENTIAL C-FOS EXPRESSION IN GABAERGIC NEURONS OF THE NUCLEUS PONTIS ORALIS DURING WAKEFULNESS AND CARBACHOL-INDUCED ACTIVE SLEEP

Previous studies have demonstrated that GABAergic processes in the nucleus pontis oralis (NPO) play an important role in the generation and maintenance of active sleep and wakefulness. However, the location of cell bodies and neural activity of this pontine GABAergic system during wakefulness and active sleep are unknown. Accordingly, the present study was undertaken to determine the distribution of activated GABAergic neurons within the NPO during wakefulness and carbachol-induced active sleep (active sleep-carbachol) using c-Fos immunocytochemistry as a functional marker of neuronal activity. Adult cats that were prepared for monitoring behavioral states were used in this experiment. In the awake group, animals were kept awake for 2 hours following the microinjection of saline into the NPO before being euthanized. In the active sleep-carbachol group, animals were euthanized after prolonged episodes of active sleep that was induced by the microinjection of carbachol into the NPO. Subsequently, brainstem sections were immunostained with a GABA antiserum in addition to a Fos polyclonal antiserum in order to double-label neurons that were both GABAergic and Fos immunoreactive. In awake cats, there was a large number of GABAedrgic neurons that expressed c-Fos in the NPO. These GABA+/c-Fos+ neurons were small to medium-sized and were located mainly within the lateral part of the NPO. In contrast, in active sleep-carbachol cats, the numbers of c-Fos expressing GABAergic neurons were decreased in the NPO. Specifically, there were only few GABAergic neurons in the lateral part of the NPO that expressed c-Fos during active sleep-carbachol. The present results demonstrate that there exists a discrete group of GABAergic neurons in the NPO that are activated during wakefulness and inactivated during active sleep. We therefore suggest that these local GABAergic cells may be the neurons that comprise the pontine GABAergic system which functions to promote wakefulness and suppress active sleep by inhibiting active sleep-generator neurons in the NPO during wakefulness and disinhibiting them during active sleep. NS092383.

The 5-HT6 receptor antagonist idalopirdine potentiates the effects of donepezil on gamma oscillations in the frontal cortex of anesthetized and awake rats without affecting sleep-wake architecture

The 5-HT6 receptor is a promising target for cognitive disorders, in particular for Alzheimer's disease (AD). The high affinity and selective 5-HT6 receptor antagonist idalopirdine (Lu AE58054) is currently in development for mild-moderate AD as adjunct therapy to acetylcholinesterase inhibitors (AChEIs). We studied the effects of idalopirdine alone and in combination with the AChEI donepezil on cortical function using two in vivo electrophysiological methods. Neuronal network oscillations in the frontal cortex were measured during electrical stimulation of the brainstem nucleus pontis oralis (nPO) in the anesthetized rat and by an electroencephalogram (EEG) in the awake, freely moving rat. In conjunction with the EEG study, we investigated the effects of idalopirdine and donepezil on sleep-wake architecture using telemetric polysomnography. Idalopirdine (2 mg/kg i.v.) increased gamma power in the medial prefrontal cortex (mPFC) during nPO stimulation. Donepezil (0.3 and 1 mg/kg i.v.) also increased cortical gamma power and pretreatment with idalopirdine (2 mg/kg i.v.) potentiated and prolonged the effects of donepezil. Similarly, donepezil (1 and 3 mg/kg s.c.) dose-dependently increased frontal cortical gamma power in the freely moving rat and pretreatment with idalopirdine (10 mg/kg p.o.) augmented the effect of donepezil 1 mg/kg. Analysis of the sleep-wake architecture showed that donepezil (1 and 3 mg/kg s.c.) dose-dependently delayed sleep onset and decreased the time spent in both REM and non REM sleep stages. In contrast, idalopirdine (10 mg/kg p.o.) did not affect sleep-wake architecture nor the effects of donepezil. In summary, we show that idalopirdine potentiates the effects of donepezil on frontal cortical gamma oscillations, a pharmacodynamic biomarker associated with cognition, without modifying the effects of donepezil on sleep. The increased cortical excitability may contribute to the procognitive effects of idalopirdine in donepezil-treated AD patients.

The 5-HT6 receptor antagonist idalopirdine potentiates the effects of acetylcholinesterase inhibition on neuronal network oscillations and extracellular acetylcholine levels in the rat dorsal hippocampus

The 5-HT6 receptor has emerged as a promising target for cognitive disorders and combining a 5-HT6 receptor antagonist with an acetylcholinesterase inhibitor (AChEI) represents a novel approach for the symptomatic treatment of Alzheimer's disease (AD). A recent phase 2 trial showed that the selective 5-HT6 receptor antagonist idalopirdine (Lu AE58054) improved cognition in patients with moderate AD on stable treatment with the AChEI donepezil. Here we investigated the effects of idalopirdine in combination with donepezil on hippocampal function using in vivo electrophysiology and microdialysis. Network oscillations in the hippocampus were recorded during electrical stimulation of the brainstem nucleus pontis oralis (nPO) in the anesthetized rat and hippocampal acetylcholine (ACh) levels were measured in the freely-moving rat. In addition, potential pharmacokinetic interactions between idalopirdine and donepezil were assessed. Idalopirdine alone did not affect hippocampal network oscillations or ACh levels. Donepezil (0.3 and 1.0 mg/kg i.v.) dose-dependently increased hippocampal theta and gamma power during nPO stimulation. Idalopirdine (2 mg/kg i.v.), administered 1 h prior to donepezil, potentiated the theta and gamma response to 0.3 mg/kg donepezil and prolonged the gamma response to 1 mg/kg donepezil. Donepezil (1.3 mg/kg s.c.) increased extracellular ACh levels in the hippocampus and this was further augmented by administration of idalopirdine (10 mg/kg p.o.) 2 h prior to donepezil. These effects could not be attributed to a pharmacokinetic interaction between the compounds. This study demonstrates that idalopirdine potentiates the effects of donepezil on two pharmacodynamic biomarkers associated with cognition, i.e. neuronal oscillations and extracellular ACh levels in the hippocampus. Such potentiation could contribute to the procognitive effects of idalopirdine observed in donepezil-treated AD patients.

Neocortical 40Hz oscillations during carbachol-induced rapid eye movement sleep and cataplexy.

Higher cognitive functions require the integration and coordination of large populations of neurons in cortical and subcortical regions. Oscillations in the gamma band (30-45Hz) of the electroencephalogram (EEG) have been involved in these cognitive functions. In previous studies, we analysed the extent of functional connectivity between cortical areas employing the 'mean squared coherence' analysis of the EEG gamma band. We demonstrated that gamma coherence is maximal during alert wakefulness and is almost absent during rapid eye movement (REM) sleep. The nucleus pontis oralis (NPO) is critical for REM sleep generation. The NPO is considered to exert executive control over the initiation and maintenance of REM sleep. In the cat, depending on the previous state of the animal, a single microinjection of carbachol (a cholinergic agonist) into the NPO can produce either REM sleep [REM sleep induced by carbachol (REMc)] or a waking state with muscle atonia, i.e. cataplexy [cataplexy induced by carbachol (CA)]. In the present study, in cats that were implanted with electrodes in different cortical areas to record polysomnographic activity, we compared the degree of gamma (30-45Hz) coherence during REMc, CA and naturally-occurring behavioural states. Gamma coherence was maximal during CA and alert wakefulness. In contrast, gamma coherence was almost absent during REMc as in naturally-occurring REM sleep. We conclude that, in spite of the presence of somatic muscle paralysis, there are remarkable differences in cortical activity between REMc and CA, which confirm that EEG gamma (≈40Hz) coherence is a trait that differentiates wakefulness from REM sleep.

Neuronal network activity in the hippocampus of tau transgenic (Tg4510) mice

Development of neurofibrillary tangles in Alzheimer's disease correlates with neuronal loss and dementia. Transgenic Tg4510 mice model the tauopathy of the disease, with these mice exhibiting progressive, region-specific neuronal loss, and behavioral deficits. In the present study, neuronal network activity in the hippocampus of 7-month-old Tg4510 mice was investigated and compared with age-matched wild-type (WT) mice. Multisite field potentials were recorded using 16-site silicon probes, inserted across the hippocampus in urethane anesthetized mice. The hippocampal network theta oscillation was evaluated in these mice by stimulating the brainstem nucleus pontis oralis. Subsequently, population spikes in the dentate gyrus were identified in response to perforant path stimulation, and long-term potentiation was elicited by theta burst stimulation. Tg4510 mice showed dramatically reduced dentate gyrus population spike amplitude; however, the magnitude of theta burst stimulation-induced long-term potentiation was identical in WT and transgenic mice. WT and Tg4510 mice showed identical increase in frequency to nucleus pontis oralis stimulation, whereas absolute theta power was severely reduced in the Tg4510 animals. Because total signal power over the entire frequency band range was reduced, there was no difference in relative theta power between WT and Tg4510 mice. These presently described electrophysiological findings can be directly attributed to the drastic reduction of pyramidal/granule neurons in Tg4510 mice, which could be the main contributing factor to their impaired behavior and cognitive function. However, the remaining synapses and neuronal circuitry seem to function properly in these assays.

Nicotinic receptor excitation of descending pain modulatory pathways

Synchronization of neuronal network oscillations within the cortex and hippocampus has been closely linked to various cognitive domains, including attention, learning, and memory. The frequency, power, and connectivity of hippocampal oscillations provide quantitative measures for examining the modulation of network activity, which influences mnemonic functions and memory formation. The wide distribution of α7 nicotinic acetylcholine receptors (α7 nAChRs) throughout the hippocampus makes them well positioned to modulate neuronal network activity. Elicitation of hippocampal theta through high frequency stimulation of the brainstem nucleus pontis oralis (nPO) is shown to be sensitive to several agents exhibiting pharmacological effects on cognition, thus representing a suitable preclinical screening assay for such drugs, including α7 nAChR agonists. We hypothesize that increases in theta power and theta-phase gamma-amplitude coupling due to α7 nAChR agonists during elicited hippocampal oscillations could reflect changes in synchronous activity of pyramidal neurons which is a critical factor for hippocampal-dependent cognitive function. In this review, four major topics are discussed: neuronal network oscillations in the hippocampus, the characteristics and distribution of α7 nAChRs therein, the modulation of elicited hippocampal theta and gamma oscillations by α7 nAChR agonists, as well as potential intrinsic roles of α7 nAChRs in hippocampal oscillations using α7 nAChR knock-out mice.

Heart rate variability during carbachol-induced REM sleep and cataplexy

The nucleus pontis oralis (NPO) exerts an executive control over REM sleep. Cholinergic input to the NPO is critical for REM sleep generation. In the cat, a single microinjection of carbachol (a cholinergic agonist) into the NPO produces either REM sleep (REMc) or wakefulness with muscle atonia (cataplexy, CA).In order to study the central control of the heart rate variability (HRV) during sleep, we conducted polysomnographic and electrocardiogram recordings from chronically prepared cats during REMc, CA as well as during sleep and wakefulness. Subsequently, we performed statistical and spectral analyses of the HRV.The heart rate was greater during CA compared to REMc, NREM or REM sleep. Spectral analysis revealed that the low frequency band (LF) power was significantly higher during REM sleep in comparison to REMc and CA. Furthermore, we found that during CA there was a decrease in coupling between the RR intervals plot (tachogram) and respiratory activity. In contrast, compared to natural behavioral states, during REMc and CA there were no significant differences in the HRV based upon the standard deviation of normal RR intervals (SDNN) and the mean squared difference of successive intervals (rMSSD).In conclusion, there were differences in the HRV during naturally-occurring REM sleep compared to REMc. In addition, in spite of the same muscle atonia, the HRV was different during REMc and CA. Therefore, the neuronal network that controls the HRV during REM sleep can be dissociated from the one that generates the muscle atonia during this state.

Interactions between hypocretinergic and GABAergic systems in the control of activity of neurons in the cat pontine reticular formation

Anatomical studies have demonstrated that hypocretinergic and GABAergic neurons innervate cells in the nucleus pontis oralis (NPO), a nucleus responsible for the generation of active (rapid eye movement (REM)) sleep (AS) and wakefulness (W). Behavioral and electrophysiological studies have shown that hypocretinergic and GABAergic processes in the NPO are involved in the generation of AS as well as W. An increase in hypocretin in the NPO is associated with both AS and W, whereas GABA levels in the NPO are elevated during W. We therefore examined the manner in which GABA modulates NPO neuronal responses to hypocretin. We hypothesized that interactions between the hypocretinergic and GABAergic systems in the NPO play an important role in determining the occurrence of AS or W. To determine the veracity of this hypothesis, we examined the effects of the juxtacellular application of hypocretin-1 and GABA on the activity of NPO neurons, which were recorded intracellularly, in chloralose-anesthetized cats. The juxtacellular application of hypocretin-1 significantly increased the mean amplitude of spontaneous EPSPs and the frequency of discharge of NPO neurons; in contrast, the juxtacellular microinjection of GABA produced the opposite effects, i.e., there was a significant reduction in the mean amplitude of spontaneous EPSPs and a decrease in the discharge of these cells. When hypocretin-1 and GABA were applied simultaneously, the inhibitory effect of GABA on the activity of NPO neurons was reduced or completely blocked. In addition, hypocretin-1 also blocked GABAergic inhibition of EPSPs evoked by stimulation of the laterodorsal tegmental nucleus. These data indicate that hypocretin and GABA function within the context of a neuronal gate that controls the activity of AS-on neurons. Therefore, we suggest that the occurrence of either AS or W depends upon interactions between hypocretinergic and GABAergic processes as well as inputs from other sites that project to AS-on neurons in the NPO.

GABAA receptors are located in cholinergic terminals in the nucleus pontis oralis of the rat: Implications for REM sleep control

The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as a rapid eye movement (REM) sleep induction zone. Microinjection of GABAA receptor antagonists into PnO induces a long lasting increase in REM sleep, which is similar to that produced by cholinergic agonists. We previously showed that this REM sleep-induction can be completely blocked by a muscarinic antagonist, indicating that the REM sleep-inducing effect of GABAA receptor antagonism is dependent upon the local cholinergic system. Consistent with these findings, it has been reported that GABAA receptor antagonists microdialyzed into PnO resulted in increased levels of acetylcholine. We hypothesize that GABAA receptors located on cholinergic boutons in the PnO are responsible for the REM sleep induction by GABAA receptor antagonists through blocking GABA inhibition of acetylcholine release. Cholinergic, varicose axon fibers were studied in the PnO by immunofluorescence and confocal, laser scanning microscopy. Immunoreactive cholinergic boutons were found to be colocalized with GABAA receptor subunit protein γ2. This finding implicates a specific subtype and location of GABAA receptors in PnO of rat in the control of REM sleep.

GABAA receptors implicated in REM sleep control express a benzodiazepine binding site

It has been reported that non-subtype-selective GABAA receptor antagonists injected into the nucleus pontis oralis (PnO) of rats induced long-lasting increases in REM sleep. Characteristics of these REM sleep increases were identical to those resulting from injection of muscarinic cholinergic agonists. Both actions were blocked by the muscarinic antagonist, atropine. Microdialysis of GABAA receptor antagonists into the PnO resulted in increased acetylcholine levels. These findings were consistent with GABAA receptor antagonists disinhibiting acetylcholine release in the PnO to result in an acetylcholine-mediated REM sleep induction. Direct evidence has been lacking for localization in the PnO of the specific GABAA receptor-subtypes mediating the REM sleep effects. Here, we demonstrated a dose-related, long-lasting increase in REM sleep following injection (60nl) in the PnO of the inverse benzodiazepine agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline (DMCM, 10−2M). REM sleep increases were greater and more consistently produced than with the non-selective antagonist gabazine, and both were blocked by atropine. Fluorescence immunohistochemistry and laser scanning confocal microscopy, colocalized in PnO vesicular acetylcholine transporter, a presynaptic marker of cholinergic boutons, with the γ2 subunit of the GABAA receptor. These data provide support for the direct action of GABA on mechanisms of acetylcholine release in the PnO. The presence of the γ2 subunit at this locus and the REM sleep induction by DMCM are consistent with binding of benzodiazepines by a GABAA receptor-subtype in control of REM sleep.