Pontine Cholinergic Neurons Research Articles

Pontine cholinergic neurons depend on three neuroprotection systems to resist nitrosative stress

Brainstem cholinergic populations survive in neurodegenerative disease, while basal forebrain cholinergic neurons degenerate. We have postulated that variable resistance to oxidative stress may in part explain this. Rat primary cultures were used to study the effects of several nitrosative/oxidative stressors on brainstem (upper pons, containing pedunculopontine and lateraldorsal tegmental nuclei; BS) cholinergic neurons, comparing them with medial septal (MS), and striatal cholinergic neurons. BS cholinergic neurons were significantly more resistant to S-nitro- N-acetyl- d, l-penicillamine (SNAP), sodium nitroprusside (SNP), and hydrogen peroxide than were MS cholinergic neurons, which in turn were more resistant than striatal cholinergic neurons. Pharmacological analyses using specific inhibitors of neuroprotective systems also revealed differences between these three cholinergic populations with respect to their vulnerability to SNAP. Toxicity of SNAP to BS neurons was exacerbated by blocking NF-κB activation with SN50 or ERK1/2 activation by PD98059, or by inhibition of phosphoinositide-3 kinase (PI3K) activity by LY294002. In contrast, SNAP toxicity to MS neurons was augmented only by SN50, and SNAP toxicity to striatal cholinergic neurons was not increased by any of these three pharmacological agents. In neuron-enriched primary cultures, BS cholinergic neurons remained resistant to SNAP while MS cholinergic neurons remained vulnerable to this agent. Immunohistochemical experiments demonstrated nitric oxide (NO)-induced increases in nuclear levels of phospho-epitopes for ERK1/2 and Akt, and of the p65 subunit of NF-κB, within BS cholinergic neurons. These data indicate that the relative resistance of BS cholinergic neurons to toxic levels of nitric oxide involves three intrinsic neuroprotective pathways that control transcriptional and anti-apoptotic cellular functions.

Dialysis delivery of an adenosine A1 receptor agonist to the pontine reticular formation decreases acetylcholine release and increases anesthesia recovery time.

Adenosine modulates cell excitability, acetylcholine release, nociception, and sleep. Pontine cholinergic neurotransmission contributes to the generation and maintenance of electroencephalographic and behavioral arousal. Adenosine A(1) receptors inhibit arousal-promoting, pontine cholinergic neurons, and adenosine enhances sleep. No previous studies have determined whether pontine adenosine also modulates recovery from anesthesia. Therefore, the current study tested the hypotheses that dialysis delivery of the adenosine A(1) receptor agonist N6-p-sulfophenyladenosine (SPA) into the pontine reticular formation would decrease acetylcholine release and increase the time needed for recovery from halothane anesthesia. A microdialysis probe was positioned in the pontine reticular formation of halothane-anesthetized cats. Probes were perfused with Ringer's solution (control) followed by the adenosine A(1) receptor agonist SPA (0.088 or 8.8 mm). Dependent measures included acetylcholine release and a numeric assessment of recovery from anesthesia. An intensive, within-subjects design and analysis of variance evaluated SPA's main effect on acetylcholine release and anesthetic recovery. The adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 microm) was coadministered with SPA to test for antagonist blocking of SPA's effects. SPA significantly (P < 0.0001) decreased acetylcholine release in the pontine reticular formation and significantly (P < 0.0001) delayed recovery from anesthesia. Coadministration of SPA and DPCPX caused no decrease in acetylcholine release or delay in postanesthetic recovery. Dialysis delivery of SPA into the cerebellar cortex confirmed that the SPA effects were site-specific to the pontine reticular formation. The results provide a novel extension of the sleep-promoting effects of adenosine by showing that pontine delivery of an adenosine A(1) receptor agonist delays resumption of wakefulness following halothane anesthesia. This extension is consistent with a potentially larger relevance of the current findings for efforts to specify neurons and molecules causing physiologic and behavioral traits comprising anesthetic states. These data support the conclusion that adenosine A(1) receptors in medial regions of the pontine reticular formation, known to modulate sleep, also contribute to the generation and/or maintenance of halothane anesthesia.

Neurochemical perspectives on the control of breathing during sleep.

A specific depression of minute ventilation occurs during sleep in normal subjects. This sleep-related ventilatory depression is partially related to mechanical events and upper airway atonia but some data also indicate that it is likely to be centrally mediated. This paper reviews the anatomical and neurochemical connections between sleep/wake- and respiratory-related areas in an attempt to identify the potential implication of sleep-related neurochemicals (serotonin, catecholamines, GABA, acetylcholine) in the sleep-related hypoventilation. The review of available data suggests that the sleep-related ventilatory depression depends upon the enhanced GABAergic activity together with a loss of suprapontine influence depending on the cessation of activity of the reticular formation. During REM sleep, an additional inhibitory activity emerges from the pontine cholinergic neurons, which contributes to the breathing irregularities and the associated depression of minute ventilation and ventilatory response to chemical stimuli. This model may contribute to a better understanding of the neurochemical environment of respiratory neurons during sleep, which remains a question of importance regarding the numerous pathological states that are linked to specific perturbations of breathing control during sleep.

Pontine cholinergic neurons show Fos-like immunoreactivity associated with cholinergically induced REM sleep.

Recently, we showed c-fos expression in pontine nuclei in association with cholinergically induced REM sleep (REMc). Pontine cholinergic mechanisms have been implicated in the orchestration of the phasic and tonic events underlying REM sleep. Therefore, in the present study, we examined whether pontine cholinergic neurons demonstrate Fos-like immunoreactivity (Fos-LI) following cholinergically induced, sustained rapid-eye movement (REMc) sleep in cats. Microinjections (0.25 microliter) of vehicle (n = 2) or carbachol (n = 3; 2.0 micrograms/0.25 microliter) were made into the medial pontine reticular formation. Carbachol produced a state with all the signs of natural REM sleep, and with durations ranging from 27 to 40.1 min. Animals were killed immediately after the end of REMc. Compared to vehicle treated animals (0.9% saline), the animals with REMc showed a significantly higher number of Fos-LI cells in pontine regions implicated in REM sleep generation. More importantly, 11.2% (SEM +/- 0.83) of cholinergic neurons in the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei were determined to be also Fos-LI positive. In the vehicle treated animals very few Fos-LI cells were found and none of these were found to be cholinergic. These findings indicate that during REMc a transcriptional cascade involving c-fos occurs in a subpopulation of pontine cholinergic neurons.

Interleukin-1β and tumor necrosis factor-α co-operatively enhance a novel trophic activity of astrocytes for pontine cholinergic neurons in vitro

Insulin and insulin-like growth factors (IGFs) are the only known trophic factors for pontine cholinergic neurons. The present study revealed that astrocyte-extract pretreated with IL-1β and TNF-α significantly enhanced choline acetyltransferase (ChAT) activity of the pontine neurons in the presence of a supramaximal dose of insulin, while various trophic factors including IGFs failed to increase the ChAT activity under the same culture conditions, suggesting that IL-1β and TNF-α co-operatively enhanced the expression of a novel trophic factor for pontine cholinergic neurons in astrocytes.

Basal magnocellular and pontine cholinergic neurons coexpress FGF receptor mRNA

By in situ hybridization histochemistry, fibroblast growth factor receptor gene ( flg)-expressing neurons were newly identified in the basal magnocellular nuclei (the vertical and horizontal limbs of the diagonal band, and Meynert's nucleus). The present study also confirmed flg localization in the laterodorsal tegmental nucleus and the pedunculopontine tegmental nucleus of the pons. Immuno- and in situ hybridization histochemistry on the same sections demonstrated that choline acetyltransferase and flg were colocalized in single neurons of the diagonal band, Meynert's nucleus and the pontine tegmental areas. The results suggest that a significant number of the basal magnocellular and a majority of the mesopontine cholinergic neurons are directly affected by fibroblast growth factors (FGF) via FGF receptor gene.

Choline acetyltransferase in schizophrenia

To test the hypothesis that schizophrenia involves altered cholinergic tone in the pons, the authors studied post-mortem brain tissue from subjects with schizophrenia. The authors used Western immunoblot to measure the concentration of choline acetyltransferase, an acetylcholine synthesizing enzyme, in the post-mortem brain tissue of 25 schizophrenic subjects and 28 nonschizophrenic comparison subjects. They also measured the concentration of glial fibrillary acidic protein, a protein from astrocytes, to examine the question of neurodegeneration. The pontine choline acetyltransferase concentrations of the schizophrenic subjects were 46% lower than those of comparison subjects, a significant difference. Glial fibrillary acidic protein concentrations did not differ between the two groups. The lower concentration of choline acetyltransferase in the pontine tegmentum of schizophrenic subjects compared with comparison subjects suggests involvement of pontine cholinergic neurons in schizophrenia.

Pontine cholinergic neurons simultaneously innervate two thalamic targets

Cholinergic neurons located in the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei have been shown to principally innervate the thalamus. In order to determine whether some of these neurons might simultaneously project to two thalamic targets we made microinjections of rhodamine-conjugated microbeads into the central-lateral nucleus of the thalamus and fluorescein isothiocynate (FITC)-conjugated microbeads into the dorso-lateral geniculate nucleus. We then determined whether both tracers were found in immunohistochemically identified cholinergic somata in the LDT and PPT nuclei. Results showed that some cholinergic and non-cholinergic neurons in the LDT and PPT nuclei projected to both thalamic sites. This finding extends our understanding of the projections of the LDT-PPT cholinergic neurons and further supports the role of these neurons in complex behaviors.

Localization of FGF receptor mRNA in the adult rat central nervous system by in situ hybridization

Fibroblast growth factor receptor (FGF-R) mRNA expression was examined in the adult rat CNS. Northern blot analysis showed a distinct 4.3 kb transcript in various CNS regions. In situ hybridization revealed widely distributed, but specific, populations of cells that express FGF-R mRNA. The most intense hybridization signals were observed in the hippocampus and in the pontine cholinergic neurons. The limbic system and brainstem nuclei, including motor nuclei, showed robust labeling. Cerebellar granule cells and spinal cord neurons were positive for FGF-R mRNA. The distribution of FGF-R mRNA differed significantly from that of NGF receptor mRNA; particularly, no hybridization signal was detected in basal forebrain cholinergic neurons. These results strongly suggest that FGF or FGF-like molecules may exert effects on specific neuronal populations in the mature CNS.

The effect of concussive head injury on central cholinergic neurons

This study examined the effect of fluid percussion head injury on the activity of cholinergic neurons in specific brain areas of the rat 12 min, 4 h and 24 h following injury. Acetylcholine (ACh) turnover, used as an index of cholinergic neuronal activity, was determined using a gas chromatographic-mass spectrometric technique. The most striking changes in cholinergic activity were observed in the dorsal pontine tegmentum, where concussive head injury produced an increase in ACh turnover 12 min and 4 h following injury. This area has been previously associated with behavioral changes observed following concussive injury. ACh turnover in the thalamus, a region to which pontine cholinergic neurons project, also tended to increase 4 h following injury. On the other hand, ACh turnover tended to decrease in the amygdala 4 h following injury. Although there were no significant changes in hippocampal ACh content or turnover following injury, ACh content did tend to increase in that brain region 12 min following injury. There were no significant effects of injury on cholinergic neurons in the cingulate/frontal cortex. These changes in cholinergic neuronal activity may contribute to the neurological deficits following concussive injury. In particular, activation of cholinergic neurons in the pontine region may contribute to components of behavioral suppression associated with reversible traumatic unconsciousness. More generalized changes in cholinergic function may lead to the production of more chronic deficits.

Retrograde labeling of neurones in the brain stem following injections of [3H]choline into the forebrain of the rat.

In an attempt to identify cholinergic neurons of the brain stem which project to the forebrain, retrograde labeling of neurons in the brain stem was examined by autoradiography following injections of 20 microCi [3H]choline into the thalamus, hypothalamus, basal forebrain and frontal cortex. After injections into the thalamus, retrogradely labeled neurons were evident within the lateral caudal mesencephalic and dorsolateral oral pontine tegmentum (particularly in the laterodorsal and pedunculopontine tegmental nuclei) and in smaller number within the latero-medial caudal pontine (Reticularis pontis caudalis, Rpc) and medullary (Reticularis gigantocellularis, Rgc) reticular formation. Following [3H]choline injections into the lateral hypothalamus and into the basal forebrain, retrogradely labeled neurons were localized in the dorsolateral caudal midbrain and oral pontine tegmentum and in smaller number in the medial medullary reticular formation (Rgc), as well as in the midbrain, pontine and medullary raphe nuclei. After injections into the anterior medial frontal cortex, a small number of retrogradely labeled cells were found in the brain stem within the laterodorsal tegmental nucleus and the dorsal raphe nucleus. In a parallel immunohistochemical study, choline acetyltransferase (ChAT)-positive neurons were found to be located in most of the regions of the reticular formation where cells were retrogradely labeled from the forebrain following [3H]choline injections. These results suggest that multiple cholinergic neurons within the lateral caudal midbrain and dorsolateral oral pontine tegmentum and a few within the caudal pontine and medullary reticular formation project to the thalamus, hypothalamus and basal forebrain and that a limited number of pontine cholinergic neurons project to the frontal cortex.