Cross-frequency coupling between slow harmonics via the real brainstem oscillators: An in vivo animal study.

Brain waves of discrete rhythms (gamma to delta frequency ranges) are ubiquitously recorded and interpreted with respect to probable corresponding specific functions. The most challenging idea of interpreting varied frequencies of brain waves has been postulated as a communication mechanism in which different neuronal assemblies use specific ranges of frequencies cooperatively. One promising candidate is cross-frequency coupling (CFC), in which some neuronal assemblies efficiently utilize the fastest gamma range brain waves as an information carrier (phase-amplitude CFC); however, phase-phase CFC via the slowest delta and theta waves has rarely been described to date. Moreover, CFC has rarely been reported in the animal brainstem including humans, which most likely utilizes the slowest waves (delta and theta ranges). Harmonic waves are characterized by the presence of a fundamental frequency with several overtones, multiples of the fundamental frequency. Rat brainstem waves seemed to consist of slow harmonics with different frequencies that could cooperatively produce a phase-phase CFC. Harmonic rhythms of different frequency ranges can cross-couple with each other to sustain robust and resilient consonance via real oscillators, notwithstanding any perturbations.

Cooperative Phase Adaptation and Amplitude Amplification of Neuronal Activity in the Vagal Complex: An Interplay Between Microcircuits and Macrocircuits.

Clusters of neurons can communicate with others through the cross-frequency coupling mechanism of oscillatory synchrony. We addressed the hypothesis that neuronal networks at various levels from micro- to macrocircuits implement this communication strategy. An abundance of local recurrent axons of vagal complex (VC) cells establish dense local microcircuits and seem to generate high-frequency noise-causing stochastic resonance (reverberation) and coherence resonance, even in in vitro slice preparations. These phenomena were observed in vitro as the generation of episodes of higher-frequency noise after an external stimulation and as stimulus-induced or spontaneous high-amplitude signals (postsynaptic activities). The in vitro microcircuit networks rarely sustained the stochastic resonance and coherence resonance cooperatively; however, in vivo networks involving additional intrabulbar mesocircuits and large-scale macrocircuits were able to sustain them cooperatively. This gave rise to large-scale oscillatory synchrony leading to robust power and coherence of signals with high amplitudes, reaching several millivolts in amplitude from a noise level of ~100 microV through cardiorespiratory frequency coupling. A regenerative mechanism of neuronal circuits might work for the generation of large-scale oscillatory synchrony. The amplitude and phase of neuronal activity in vivo may interact cooperatively to give rise to varying degrees of power and coherence of robust rhythmic activity for distinct physiological roles. The cooperative interaction between phase adaptation and amplitude amplification of neuronal activity may provide diverse nervous systems with both robustness and resilience.

Differential Ascending Projections From the Male Rat Caudal Nucleus of the Tractus Solitarius: An Interface Between Local Microcircuits and Global Macrocircuits.

To integrate and broadcast neural information, local microcircuits and global macrocircuits interact within certain specific nuclei of the central nervous system. The structural and functional architecture of this interaction was determined for the caudal nucleus of the tractus solitarius (NTS) at the level of the area postrema (AP), a relay station of peripheral viscerosensory information that is processed and conveyed to brain regions concerned with autonomic-affective and other interoceptive reflexive functions. Axon collaterals of most small NTS cells (soma <150 µm2) establish excitatory or inhibitory local microcircuits likely to control the activity of nearby NTS cells and to transfer peripheral signals to efferent projection neurons. At least two types of cells that constitute efferent pathways from the caudal NTS (cNTS) were distinguished: (1) a greater numbers of small cells, seemingly forming local excitatory microcircuits via recurrent axon collaterals, that project specifically and unidirectionally to the lateral parabrachial nucleus; and (2) a much smaller numbers of cells likely to establish multiple global connections, mostly via the medial forebrain bundle (MFB) or the dorsal longitudinal fascicle (DLF), with a wide range of brain regions, including the ventrolateral medulla (VLM), hypothalamus, central nucleus of the amygdala (ACe), bed nucleus of the stria terminalis (BNST), spinal cord dorsal horn, brainstem reticular formation, locus coeruleus (LC), periaqueductal gray (PAG) and periventricular diencephalon (including the epithalamus). The evidence presented here suggests that distinct cNTS cell types distinguished by projection pattern and related structural and functional features participate differentially in the computation of viscerosensory information and coordination of global macro-networks in a highly organized manner.

Spatiotemporal Structure and Dynamics of Spontaneous Oscillatory Synchrony in the Vagal Complex.

Fundamental structure and dynamics of spontaneous neuronal activities without apparent peripheral inputs were analyzed in the vagal complex (VC), whose activities had been generally thought to be produced almost passively to peripheral cues. The analysis included the caudal nucleus of the tractus solitarius-a main gateway for viscerosensory peripheral afferents and involved dynamically and critically in cardiorespiratory brainstem networks. In the present study, a possibility of self-organized brain activity was addressed in the VC. While VC neurons exhibited sparse firing in anesthetized rats and in in vitro preparations, we identified peculiar features of the emergent electrical population activity: (1) Spontaneous neuronal activity, in most cases, comprised both respiration and cardiac cycle components. (2) Population potentials of polyphasic high amplitudes reaching several millivolts emerged in synchrony with the inspiratory phase of respiratory cycles and exhibited several other characteristic temporal dynamics. (3) The spatiotemporal dynamics of local field potentials (LFPs), recorded simultaneously over multiple sites, were characterized by a stochastic emergence of high-amplitude synchrony. By adjusting amplitude and frequency (phase) over both space and time, the traveling synchrony exhibited varied degrees of coherence and power with a fluctuating balance between mutual oscillators of respiratory and cardiac frequency ranges. Full-fledged large-scale oscillatory synchrony over a wide region of the VC emerged after achieving a maximal stable balance between the two oscillators. Distinct somatic (respiratory; ~1 Hz) and visceral (autonomic; ~5 Hz) oscillators seemed to exist and communicate co-operatively in the brainstem network. Fluctuating oscillatory coupling may reflect varied degrees of synchrony influenced by the varied amplitude and frequency of neuronal activity in the VC. Intranuclear micro-, intrabulbar meso-, and wide-ranging macro-circuits involving the VC are likely to form nested networks and strategically interact to maintain a malleable whole-body homeostasis. These two brainstem oscillators could orchestrate neuronal activities of the VC, and other neuronal groups, through a phase-phase coupling mechanism to perform specific physiological functions.

Geometric and functional architecture of visceral sensory microcircuitry.

Is microcircuit wiring designed deterministically or probabilistically? Does geometric architecture predict functional dynamics of a given neuronal microcircuit? These questions were addressed in the visceral sensory microcircuit of the caudal nucleus of the tractus solitarius (NTS), which is generally thought to be homogeneous rather than laminar in cytoarchitecture. Using in situ hybridization histochemistry and whole-cell patch clamp recordings followed by neuronal reconstruction with biocytin filling, anatomical and functional organization of NTS microcircuitry was quantified to determine associative relationships. Morphologic and chemical features of NTS neurons displayed different patterns of process arborization and sub-nuclear localization according to neuronal types: smaller cells featured presynaptic local axons and GABAergic cells were aggregated specifically within the ventral NTS. The results suggested both a laminar organization and a spatial heterogeneity of NTS microcircuit connectivity. Geometric analysis of pre- and postsynaptic axodendritic arbor overlap of reconstructed neurons (according to parent somal distance) confirmed a heterogeneity of microcircuit connectivity that could underlie differential functional dynamics along the dorsoventral axis. Functional dynamics in terms of spontaneous and evoked postsynaptic current patterns behaved in a strongly location-specific manner according to the geometric dimension, suggesting a spatial laminar segregation of neuronal populations: a dorsal group of high excitation and a ventral group of balanced excitation and inhibition. Recurrent polysynaptic activity was also noted in a subpopulation of the ventral group. Such geometric and functional laminar organization seems to provide the NTS microcircuit with both reverberation capability and a differentiated projection system for appropriate computation of visceral sensory information.

Postnatal development of axosomatic synapses in the rat nucleus tractus solitarius: dorsal and ventral subnuclei differences.

Inhibitory axosomatic synapses could effectively suppress the excitability of postsynaptic cells. It is important to examine the development of inhibitory axosomatic synapses to understand the maturation of information processing. The caudal nucleus tractus solitarius (cNTS), which regulates the autonomic system, consists of several subnuclei. In the present study, development of axosomatic synapses in the dorsal and ventral subnuclei was examined by electron microscopy. In dorsal subnuclei, the percentage of GAD-positive terminals on the somata, the percentage of small cell somata with synapses and axosomatic synapse density drastically decreased from postnatal day (P) 5 to P10. In ventral subnuclei, the percentage of GAD-positive terminals on the soma, the percentage of small or large cell somata with synapses and axosomatic synapse density were maintained or increased from P5 to P10. Thus, decrease of inhibitory axosomatic synapses in dorsal subnuclei might facilitate maturation of fine receptive areas for peripheral inputs, while increase of inhibitory axosomatic synapses in ventral subnuclei might facilitate the establishment of an effective regulation system for cNTS output.

Quantitative and immunohistochemical analysis of neuronal types in the mouse caudal nucleus tractus solitarius: focus on GABAergic neurons.

gamma-Aminobutyric acid-ergic (GABAergic) neurons are major inhibitory interneurons that are widely distributed in the central nervous system. The caudal nucleus tractus solitarius (cNTS), which plays a key role in respiratory, cardiovascular, and gastrointestinal function, contains GABAergic neurons for regulation of neuronal firing. In the present study, GABAergic neuronal organization was analyzed in relation to the location of subnuclei in the mouse cNTS. According to the differential expression of glutamate decarboxylase 67 (GAD67), vesicular glutamate transporter 2 (VGLUT2), calbindin, and tyrosine hydroxylase (TH) mRNAs, the cNTS was divided into four subnuclei: the subpostrema, dorsomedial, commissural, and medial subnuclei. The numerical density and size of soma in the four subnuclei were then quantified by an unbiased dissector analysis. Calbindin-positive cells constituted subpopulations of small non-GABAergic neurons preferentially localized in the subpostrema subnucleus. TH-positive cells constituted large neurons preferentially localized in the medial subnucleus. GABAergic neurons constituted a subpopulation of small neurons, preferentially localized in the commissural and medial subnuclei, which represented > or =50% of small cells in these subnuclei. Thus, the GABAergic small neurons were located around TH-positive large cells in the ventrolateral portion of the cNTS. This finding, in combination with results of previous studies in the rat cNTS showing that large cells originate efferents from the cNTS, suggests that GABAergic small neurons in the commissural and medial subnuclei might regulate output from the cNTS.

Glial coverage of the small cell somata in the rat nucleus of tractus solitarius during postnatal development.

Astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. Previous studies suggested that axosomatic synapses decreased in number on the small cells of the rat caudal nucleus of tractus solitarius (cNTS) toward the end of the first postnatal week. Astrocytes might be involved in this phenomenon. We examined the morphological development of astrocytic processes around the small cell soma in the rat cNTS using light and electron microscopy. Glial fibrillary acidic protein (GFAP), glutamate-aspartate transporter (GLAST), and glutamate transporter-1 (GLT-1)-positive structures within the cNTS became more intensely stained as development proceeded. GLAST-positive structures encompassed calbindin-positive small cell somata after postnatal day 10. Electron microscopic observations indicated that astrocytic processes encompass the small cell soma, while the number of axosomatic synapses decreases as development proceeds. The timing for glial coverage of the small cell soma appears to be consistent with the decrease in axosomatic synapses on the small cells. These observations imply that astrocytes may participate actively in regulating the decrease of axosomatic synapses on small cells in the cNTS during postnatal development.

Postnatal development of GABAergic axon terminals in the rat nucleus of tractus solitarius.

The proper function of the brain depends on a precise arrangement of excitatory and inhibitory synapses. Although the caudal nucleus of tractus solitarius (cNTS) plays a pivotal role in cardiorespiratory reflexes, we know little about the formation of the local neural network in the cNTS. In the present study, we have focused on GABAergic axon terminals and investigated postnatal changes in GABAergic synaptic organizations in the rat cNTS immunocytochemically at both light and electron microscopic levels. Counting synaptic and non-synaptic GABAergic axon terminals revealed that GABAergic axon terminal number in the cNTS seemed constant until the second postnatal week and that GABAergic axon terminals were reorganized around postnatal day 10 (P10). Electron microscopic observation revealed that more than 20% GABAergic axon terminals formed axosomatic synapses at P2 to P4, but the number of GABAergic axosomatic synapse on neurons with smaller soma (smaller neurons) decreased considerably after P8. Orphan GABAergic boutons were present around somata of smaller neurons at P10, and axodendritic synapse number on thicker dendrites decreased gradually during postnatal development. These results show that GABAergic axon terminals detach from somata of smaller neurons at the second postnatal week. Such morphologic changes in axon terminals could cause changes in electrophysiological activity and might contribute to reorganization of the local network within the cNTS from neonatal to adult type. These postnatal changes in the cNTS local network might be prerequisite for the cardiorespiratory reflexes of the adult type.

Pattern differentiation of excitatory and inhibitory synaptic inputs on distinct neuronal types in the rat caudal nucleus of the tractus solitarius.

Region- and size-specific neuronal organizations of the caudal nucleus of the tractus solitarius (cNTS) were investigated, followed by analyses of excitatory and inhibitory synaptic input patterns onto specific cell types by patch clamp recordings and immunoelectron microscopy. Cell size distribution and numerical density of cNTS neurons were examined in subregions at levels of the area postrema. In the subpostremal and dorsomedial subnuclei, characterized by the presence of dense glutamatergic and sparse GABAergic somata, small calbindin neurons constituted 42% of the total cells. The medial subnucleus contained large numbers of glutamatergic, GABAergic, and catecholaminergic somata and large tyrosine hydroxylase-containing cells constituted 13% in this region. In total, small neurons (<150 microm2) represented about 80% of the cell population in the cNTS. Predominant excitatory postsynaptic currents were observed in the adult small neurons, while inhibitory postsynaptic currents were more evident in larger neurons, irrespective of subnuclear location. This distinct differentiation of postsynaptic current patterns was not evident in neonates. GABAergic synapses were more frequently associated with dendrites of large catecholaminergic cells (73%) than with those of small calbindin-containing cells (10%) in adults. These results indicate that differential synaptic input patterns were developmentally established in distinct small and large neurons.

Expression of RAB27B is up-regulated in senescent human cells.

Immortal SVts8 cells that express thermolabile SV40 T antigen exhibit a senescence-like phenomenon upon inactivation of the T antigen. By using a cDNA subtractive hybridization technique, RAB27B, a member of the RAB GTPase family, was found to be up-regulated in senescent SVts8 cells. The up-regulation of RAB27B depends on the p53 gene. Enhanced expression was also observed in replicative senescence in normal human fibroblasts.

Local axonal arborization patterns of distinct neuronal types in the caudal nucleus of the tractus solitarius.

Neurons in the caudal nucleus of the tractus solitarius (cNTS) are quite heterogeneous in cell size (50 to 450 microm(2) in somal area) and other morphologic characteristics. For a more objective classification of cNTS neurons, their morphologic features were analyzed quantitatively based on reconstructed biocytin-filled cells after whole-cell patch-clamp recordings. According to the patterns of axonal branching behaviors, cNTS cells could be classified into two groups: smaller cells (94.1 microm(2) in mean somal area, range 62-120 microm(2), n = 22) and larger cells (245 microm(2) in mean somal area, range 142-411 microm(2), n = 23). Extensive axonal arborization with numerous possible synaptic boutons was specifically associated with smaller neurons, while larger cells possessed no or few axon collaterals, suggesting their distinct roles as local circuit neurons (or interneurons) and projection neurons, respectively. With regard to somatodendritic characteristics, the following correlations with cell size were found: smaller cells had larger form factors than larger cells (P < 0.05). Larger neurons had more extensive dendritic arborization, expressed by total dendritic length (P < 0.01) and number of dendritic branching points (P < 0.01), than smaller cells. It was suggested that small cNTS neurons contribute specifically to an integration of input information generated in the local circuits, while large neurons convey the integrated information to other autonomic brain regions.