Electron microscopic localization of M2-muscarinic receptors in cholinergic and noncholinergic neurons of the laterodorsal tegmental and pedunculopontine nuclei of the rat mesopontine tegmentum.

Muscarinic m2 receptors (M2Rs) are implicated in autoregulatory control of cholinergic output neurons located within the pedunculopontine (PPT) and laterodorsal tegmental (LTD) nuclei of the mesopontine tegmentum (MPT). However, these nuclei contain many noncholinergic neurons in which activation of M2R heteroceptors may contribute significantly to the decisive role of the LTD and PPT in sleep-wakefulness. We examined the electron microscopic dual immunolabeling of M2Rs and the vesicular acetylcholine transporter (VAchT) in the MPT of rat brain to identify the potential sites for M2R activation. M2R immunogold labeling was predominately seen in somatodendritic profiles throughout the PPT/LTD complex. In somata, M2R immunogold particles were often associated with Golgi lamellae and cytoplasmic endomembrannes, but were rarely in contact with the plasma membrane, as was commonly seen in dendrites. Approximately 36% of the M2R-labeled somata and 16% of the more numerous M2R-labeled dendrites coexpressed VAchT. M2R and M2R/VAchT-labeled dendritic profiles received synapses from inhibitory- and excitatory-type axon terminals, over 88% of which were unlabeled and others contained exclusively M2R or VAchT immunoreactivity. In axonal profiles M2R immunogold was localized to plasmalemmal and cytoplasmic regions and showed a similar distribution in many VAchT-negative glial profiles. These results provide ultrastructural evidence suggestive of somatic endomembrane trafficking of M2Rs, whose activation serves to regulate the postsynaptic excitatory and inhibitory responses in dendrites of cholinergic and noncholinergic neurons in the MPT. They also suggest the possibility that M2Rs in this brain region mediate the effects of acetylcholine on the release of other neurotransmitters and on glial signaling. J. Comp. Neurol. 524:3084-3103, 2016. © 2016 Wiley Periodicals, Inc.

[Organization of the projections of the substructures of deep mesencephalic nucleus to the striatum in the dog brain].

Method of retrograde axonal transport of horseradish peroxidase was used to study the organization of the projections of the individual substructures of the deep mesencephalic nucleus complex to the functionally diverse regions of striatal structures of the dog brain (n=20). It was shown that the projection fibers of the neurons of the nucleus profundus mesencephali were directed to the ventro-lateral segment of the nucleus caudatus and the ventral segment of the putamen. The dorsal segment of the putamen obtains the fibers from the neurons of the nucleus cuneiformis, while the lateral segment of the nucleus accumbens received them from the neurons of the nucleus subcuneiformis. The possible pathways for conduction of the functionally diverse information and its integration in the investigated projection systems are discussed.

Distinct types of non-cholinergic pedunculopontine neurons are differentially modulated during global brain states.

The pedunculopontine nucleus (PPN) is critically involved in brain-state transitions that promote neocortical activation. In addition, the PPN is involved in the control of several behavioral processes including locomotion, motivation and reward, but the neuronal substrates that underlie such an array of functions remain elusive. Here we analyzed the physiological properties of non-cholinergic PPN neurons in vivo across distinct brain states, and correlated these with their morphological properties after juxtacellular labeling. We show that non-cholinergic neurons in the PPN whose firing is not strongly correlated to neocortical activity are highly heterogeneous and are composed of at least three different subtypes: (1) "quiescent" neurons, which are nearly silent during slow-wave activity (SWA) but respond robustly to neocortical activation; (2) "tonic firing" neurons, which have a stationary firing rate that is independent of neocortical activity across different brain states; and (3) "irregular firing" neurons, which exhibit a variable level of correlation with neocortical activity. The majority of non-cholinergic neurons have an ascending axonal trajectory, with the exception of some irregular firing neurons that have descending axons. Furthermore, we observed asymmetric synaptic contacts within the PPN arising from the axon collaterals of labeled neurons, suggesting that excitatory, non-cholinergic neurons can shape the activity of neighboring cells. Our results provide the first evidence of distinct firing properties associated with non-cholinergic neuronal subtypes in the PPN, suggesting a functional heterogeneity, and support the notion of a local network assembled by projection neurons, the properties of which are likely to determine the output of the PPN in diverse behavioral contexts.

Acetylcholine innervation of the adult rat thalamus: distribution and ultrastructural features in dorsolateral geniculate, parafascicular, and reticular thalamic nuclei.

The acetylcholine (ACh) innervation of thalamus arises mainly from the brainstem pedunculopontine and laterodorsal tegmental nuclei. By using immunocytochemistry with a monoclonal antibody against whole rat choline acetyltransferase (ChAT), we quantified the distribution and characterized the ultrastructural features of these nerve terminals (axon varicosities) in the dorsolateral geniculate (DLG), parafascicular (PF), and reticular thalamic (Rt) nuclei of adult rat. The regional density of ACh innervation was the highest in PF (2.1 x 10(6) varicosities/mm(3)), followed by Rt (1.7 x 10(6)) and DLG (1.3 x 10(6)). In single thin sections, ChAT-immunostained varicosity profiles appeared comparable in shape and content in the three nuclei, but significantly larger in PF than in DLG and Rt. The number of these profiles displaying a synaptic junction was also much higher in PF than in DLG and Rt, indicating that all ChAT-immunostained varicosities in PF were synaptic, but only 39% in DLG and 33% in Rt. The hypothesis that glutamate corelease might account for the maintenance of the entirely synaptic ACh innervation in PF was refuted by the lack of colocalization of ChAT and vesicular glutamate transporter 2 (VGLUT2) in PF axon varicosities after dual immunolabeling. These data suggest that diffuse as well as synaptic transmission convey modulatory effects of the ACh input from brainstem to DLG and Rt during waking. In contrast, the entirely synaptic ACh input to PF should allow for a direct relaying of the information from brainstem, affecting basal ganglia function as well as perceptual awareness, including attention and pain perception.

Age-related changes in cholinergic neurons in the laterodorsal and the pedunculo-pontine tegmental nuclei of cats: a combined light and electron microscopic study.

In aged cats, light microscopic studies revealed significant decrease in the soma size of choline acetyltransferase (ChAT)-positive neurons in the laterodorsal and pedunculo-pontine tegmental nuclei (LDT and PPT), compared with adult control animals. In addition, a significant reduction of the total dendritic length and total dendritic segment number of ChAT-positive neurons was detected in both the LDT and PPT of aged cats. However, in contrast to the changes of soma and dendrites, no significant changes in the number of ChAT-positive neurons in aged were found comparing to that in the control cats in both the LDT and PPT; nor were there differences in the staining intensity of the somata of neurons in the adult and aged cats. Electron microscopic analysis highlighted degenerative changes in cholinergic neurons in the LDT and PPT of aged cats which included somata with intracytoplasmic vacuoles, darkened mitochondria, depletion of dendritic microtubules and severe demyelination of axons. These data indicate that profound atrophic changes occur in cholinergic systems of the LDT and PPT as a consequence of the aging process. These alterations likely reflect the cellular bases for the age-related changes in REM sleep that occur in old animals.

Neurons expressing relaxin 3/INSL 7 in the nucleus incertus respond to stress.

Relaxin 3/INSL 7 has recently been identified as a new member of the insulin/relaxin superfamily. Although it was reported to be dominantly expressed in the brain, its detailed distribution and function in the central nervous system are still obscure. In the present study we demonstrated that in the rat relaxin 3 was mainly expressed in neurons of the nucleus incertus (NI) of the median dorsal tegmental pons. Other relaxin 3-expressing neurons were scattered in the pontine raphe nucleus, the periaqueductal gray and dorsal area to the substantia nigra in the midbrain reticular formation. Relaxin 3-immunoreactive fibers projected particularly densely in the septum, hippocampus, lateral hypothalamus and intergeniculate leaflet of the thalamus. Ultrastructural examination revealed that relaxin 3 was localized in the dense-cored vesicles in the perikarya and was also observed in the synaptic terminals of axons. As almost all relaxin 3-containing neurons express corticotropin-releasing factor (CRF) type 1 receptor in the NI, we examined the response of relaxin 3 neurons to intracerebroventricular administration of CRF; 65% of relaxin 3 neurons expressed c-Fos 2 h after intracerebroventricular administration of 1 microg CRF. We then confirmed that c-Fos was induced in 60% of relaxin 3 neurons in the NI and the expression of relaxin 3 mRNA increased significantly in the NI after water-restraint stress. Collectively, these results suggest that relaxin 3 produced in the NI is released from nerve endings and is involved in the regulation of the stress response.

Colocalization of gamma-aminobutyric acid and acetylcholine in neurons in the laterodorsal and pedunculopontine tegmental nuclei in the cat: a light and electron microscopic study.

Cholinergic and gamma-aminobutyric acid (GABA) mechanisms in the dorsolateral pontomesencephalic tegmentum have been implicated in the control of active (REM) sleep and wakefulness. To determine the relationships between neurons that contain these neurotransmitters in this region of the brainstem in adult cats, combined light and electron microscopic immunocytochemical procedures were employed. Light microscopic analyses revealed that choline acetyltransferase (ChAT) and GABA immunoreactive neurons were distributed throughout the laterodorsal and pedunculopontine tegmental nuclei (LDT and PPT). Surprisingly, approximately 50% of the ChAT immunoreactive neurons in these nuclei also contained GABA. Using electron microscopic pre-embedding immunocytochemistry, GABA immunoreactivity was observed in somas, dendrites and axon terminals in both the LDT and PPT. Most of the GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites. Electron microscopic double-immunolabeling techniques revealed that ChAT and GABA were colocalized in axon terminals in the LDT/PPT. Approximately 30% of the ChAT immunoreactive terminals were also GABA immunoreactive, whereas only 6-8% of the GABA immunoreactive terminals were ChAT immunoreactive. Most of the ChAT/GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites; however, ChAT/GABA immunoreactive terminals were also observed that contacted ChAT immunoreactive dendrites. With respect to ChAT immunoreactive postsynaptic profiles, approximately 40% of the somas and 50% of the dendrites received synaptic contact from GABA immunoreactive terminals in both the LDT and PPT. These findings (a) indicate that there are fundamental interactions between cholinergic and GABAergic neurons within the LDT/PPT that play an important role in the control of active sleep and wakefulness and (b) provide an anatomical basis for the intriguing possibility that a mechanism of acetylcholine and GABA co-release from the terminals of LDT/PPT neurons is involved in the regulation of behavioral states.