Deep Diaphragmatic Breathing-Anatomical and Biomechanical Consideration.

Background: Deep diaphragmatic breathing (DDB) involves slow and fully contraction of the diaphragm with expansion of the belly during inhalation, and slow and fully contraction of the abdominal muscles with reduction of the belly during exhalation. It is the key component of the holistic mind-body exercises commonly used for patients with multimorbidity. Purpose: The purpose of this study was to re-visit and address the fundamental anatomical and biomechanical consideration of the DDB with the relevant literature. Method: Peer-reviewed publications from last the 15 years were retrieved, reviewed, and analyzed. Findings: In this article, we described the updated morphological and anatomical characteristics of the diaphragm. Then, we elucidated in a biomechanical approach how and why the DDB can work on the gastrointestinal, cardiopulmonary, and nervous systems as well as on regulating the intra-abdominopelvic pressure and mind-body interaction to coordinate the diaphragm-pelvic floor-abdominal complex for a variety of physical and physiological activities. Conclusion: Understanding of this updated DDB knowledge may help holistic healthcare professionals including holistic nurses provide better patient education and care management during the DDB or DDB-based mind-body intervention time.

Genetic divergence in the transcriptional engram of chronic alcohol abuse: A laser-capture RNA-seq study of the mouse mesocorticolimbic system.

Genetic factors that influence the transition from initial drinking to dependence remain enigmatic. Recent studies have leveraged chronic intermittent ethanol (CIE) paradigms to measure changes in brain gene expression in a single strain at 0, 8, 72 h, and even 7 days following CIE. We extend these findings using LCM RNA-seq to profile expression in 11 brain regions in two inbred strains - C57BL/6J (B6) and DBA/2J (D2) - 72 h following multiple cycles of ethanol self-administration and CIE. Linear models identified differential expression based on treatment, region, strain, or interactions with treatment. Nearly 40% of genes showed a robust effect (FDR < 0.01) of region, and hippocampus CA1, cortex, bed nucleus stria terminalis, and nucleus accumbens core had the highest number of differentially expressed genes after treatment. Another 8% of differentially expressed genes demonstrated a robust effect of strain. As expected, based on similar studies in B6, treatment had a much smaller impact on expression; only 72 genes (p < 0.01) are modulated by treatment (independent of region or strain). Strikingly, many more genes (415) show a strain-specific and largely opposite response to treatment and are enriched in processes related to RNA metabolism, transcription factor activity, and mitochondrial function. Over 3 times as many changes in gene expression were detected in D2 compared to B6, and weighted gene co-expression network analysis (WGCNA) module comparison identified more modules enriched for treatment effects in D2. Substantial strain differences exist in the temporal pattern of transcriptional neuroadaptation to CIE, and these may drive individual differences in risk of addiction following excessive alcohol consumption.

The identification and neurochemical characterization of central neurons that target parasympathetic preganglionic neurons involved in the regulation of choroidal blood flow in the rat eye using pseudorabies virus, immunolabeling and conventional pathway tracing methods.

The choroidal blood vessels of the eye provide the main vascular support to the outer retina. These blood vessels are under parasympathetic vasodilatory control via input from the pterygopalatine ganglion (PPG), which in turn receives its preganglionic input from the superior salivatory nucleus (SSN) of the hindbrain. The present study characterized the central neurons projecting to the SSN neurons innervating choroidal PPG neurons, using pathway tracing and immunolabeling. In the initial set of studies, minute injections of the Bartha strain of the retrograde transneuronal tracer pseudorabies virus (PRV) were made into choroid in rats in which the superior cervical ganglia had been excised (to prevent labeling of sympathetic circuitry). Diverse neuronal populations beyond the choroidal part of ipsilateral SSN showed transneuronal labeling, which notably included the parvocellular part of the paraventricular nucleus of the hypothalamus (PVN), the periaqueductal gray, the raphe magnus (RaM), the B3 region of the pons, A5, the nucleus of the solitary tract (NTS), the rostral ventrolateral medulla (RVLM), and the intermediate reticular nucleus of the medulla. The PRV+ neurons were located in the parts of these cell groups that are responsive to systemic blood pressure signals and involved in systemic blood pressure regulation by the sympathetic nervous system. In a second set of studies using PRV labeling, conventional pathway tracing, and immunolabeling, we found that PVN neurons projecting to SSN tended to be oxytocinergic and glutamatergic, RaM neurons projecting to SSN were serotonergic, and NTS neurons projecting to SSN were glutamatergic. Our results suggest that blood pressure and volume signals that drive sympathetic constriction of the systemic vasculature may also drive parasympathetic vasodilation of the choroidal vasculature, and may thereby contribute to choroidal baroregulation during low blood pressure.

Anti-oxidant polydatin (piceid) protects against substantia nigral motor degeneration in multiple rodent models of Parkinson's disease.

BACKGROUND: Compelling evidence suggests that inhibition of the complex I of the electron transport chain and elevated oxidative stress are the earliest events during the pathogenesis of Parkinson's disease (PD). Therefore, anti-oxidants, especially those from natural sources, hold good promise in treating PD as demonstrated mostly by the studies in rodent models. RESULTS: Herein, we determined if polydatin (piceid), a natural polyphenol, could exert anti-oxidative activity and attenuate dopaminergic neurodegeneration in three commonly used rodent models of PD. Male Sprague Dawley rats given rotenone subcutaneously for 5 weeks developed all the essential features of PD, including a strong increase in catalepsy score and a decrease in motor coordination activity, starting at 4 weeks. Selective increase in oxidative damage was found in the striatal region as compared to the hippocampus and cortex, accompanied by massive degeneration of dopaminergic neurons in the substantia nigra (SNc). Co-administration of piceid orally was able to attenuate rotenone-induced motor defects in a dose dependent manner, with 80 mg/kg dosage showing even better effect than L-levodopa (L-dopa). Piceid treatment significantly prevented the rotenone-induced changes in the levels of glutathione, thioredoxin, ATP, malondialdehyde (MDA) and the manganese superoxide dismutases (SOD) in striatum. Furthermore, piceid treatment rescued rotenone-induced dopaminergic neurodegeneration in the SNc region. Similar protective effect of piceid was also observed in two additional models of PD, MPTP in mice and 6-OHDA in rats, showing corrected motor functions, SOD and MDA activities as well as p-Akt and activated caspase-3 levels. CONCLUSION: In three rodent models of PD, piceid preserves and corrects several major anti-oxidant pathways/parameters selectively in the affected SNc region. This implies its potent anti-oxidant activity as one major underscoring mechanism for protecting the vulnerable SNc neurodegeneration in these models. Taken together, these findings strongly suggest a therapeutic potential of piceid in treating PD.

Cocaine inhibition of nicotinic acetylcholine receptors influences dopamine release.

Nicotinic acetylcholine receptors (nAChRs) potently regulate dopamine (DA) release in the striatum and alter cocaine's ability to reinforce behaviors. Since cocaine is a weak nAChR inhibitor, we hypothesized that cocaine may alter DA release by inhibiting the nAChRs in DA terminals in the striatum and thus contribute to cocaine's reinforcing properties primarily associated with the inhibition of DA transporters. We found that biologically relevant concentrations of cocaine can mildly inhibit nAChR-mediated currents in midbrain DA neurons and consequently alter DA release in the dorsal and ventral striatum. At very high concentrations, cocaine also inhibits voltage-gated Na channels in DA neurons. Furthermore, our results show that partial inhibition of nAChRs by cocaine reduces evoked DA release. This diminution of DA release via nAChR inhibition more strongly influences release evoked at low or tonic stimulation frequencies than at higher (phasic) stimulation frequencies, particularly in the dorsolateral striatum. This cocaine-induced shift favoring phasic DA release may contribute to the enhanced saliency and motivational value of cocaine-associated memories and behaviors.

Protection genes in nucleus accumbens shell affect vulnerability to nicotine self-administration across isogenic strains of adolescent rat.

Classical genetic studies show the heritability of cigarette smoking is 0.4-0.6, and that multiple genes confer susceptibility and resistance to smoking. Despite recent advances in identifying genes associated with smoking behaviors, the major source of this heritability and its impact on susceptibility and resistance are largely unknown. Operant self-administration (SA) of intravenous nicotine is an established model for smoking behavior. We recently confirmed that genetic factors exert strong control over nicotine intake in isogenic rat strains. Because the processing of afferent dopaminergic signals by nucleus accumbens shell (AcbS) is critical for acquisition and maintenance of motivated behaviors reinforced by nicotine, we hypothesized that differential basal gene expression in AcbS accounts for much of the strain-to-strain variation in nicotine SA. We therefore sequenced the transcriptome of AcbS samples obtained by laser capture microdissection from 10 isogenic adolescent rat strains and compared all RNA transcript levels with behavior. Weighted gene co-expression network analysis, a systems biology method, found 12 modules (i.e., unique sets of genes that covary across all samples) that correlated (p<0.05) with amount of self-administered nicotine; 9 of 12 correlated negatively, implying a protective role. PCR confirmed selected genes from these modules. Chilibot, a literature mining tool, identified 15 genes within 1 module that were nominally associated with cigarette smoking, thereby providing strong support for the analytical approach. This is the first report demonstrating that nicotine intake by adolescent rodents is associated with the expression of specific genes in AcbS of the mesolimbic system, which controls motivated behaviors. These findings provide new insights into genetic mechanisms that predispose or protect against tobacco addiction.

Genome-Wide Gene Expression Profiling of Nucleus Accumbens Neurons Projecting to Ventral Pallidum Using both Microarray and Transcriptome Sequencing.

The cellular heterogeneity of brain poses a particularly thorny issue in genome-wide gene expression studies. Because laser capture microdissection (LCM) enables the precise extraction of a small area of tissue, we combined LCM with neuronal track tracing to collect nucleus accumbens shell neurons that project to ventral pallidum, which are of particular interest in the study of reward and addiction. Four independent biological samples of accumbens projection neurons were obtained. Approximately 500 pg of total RNA from each sample was then amplified linearly and subjected to Affymetrix microarray and Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD) transcriptome sequencing (RNA-seq). A total of 375 million 50-bp reads were obtained from RNA-seq. Approximately 57% of these reads were mapped to the rat reference genome (Baylor 3.4/rn4). Approximately 11,000 unique RefSeq genes and 100,000 unique exons were identified from each sample. Of the unmapped reads, the quality scores were 4.74 ± 0.42 lower than the mapped reads. When RNA-seq and microarray data from the same samples were compared, Pearson correlations were between 0.764 and 0.798. The variances in data obtained for the four samples by microarray and RNA-seq were similar for medium to high abundance genes, but less among low abundance genes detected by microarray. Analysis of 34 genes by real-time polymerase chain reaction showed higher correlation with RNA-seq (0.66) than with microarray (0.46). Further analysis showed 20-30 million 50-bp reads are sufficient to provide estimates of gene expression levels comparable to those produced by microarray. In summary, this study showed that picogram quantities of total RNA obtained by LCM of ∼700 individual neurons is sufficient to take advantage of the benefits provided by the transcriptome sequencing technology, such as low background noise, high dynamic range, and high precision.

Projections from the hypothalamic paraventricular nucleus and the nucleus of the solitary tract to prechoroidal neurons in the superior salivatory nucleus: Pathways controlling rodent choroidal blood flow.

Using intrachoroidal injection of the transneuronal retrograde tracer pseudorabies virus (PRV) in rats, we previously localized preganglionic neurons in the superior salivatory nucleus (SSN) that regulate choroidal blood flow (ChBF) via projections to the pterygopalatine ganglion (PPG). In the present study, we used higher-order transneuronal retrograde labeling following intrachoroidal PRV injection to identify central neuronal cell groups involved in parasympathetic regulation of ChBF via input to the SSN. These prominently included the hypothalamic paraventricular nucleus (PVN) and the nucleus of the solitary tract (NTS), both of which are responsive to systemic BP and are involved in systemic sympathetic vasoconstriction. Conventional pathway tracing methods were then used to determine if the PVN and/or NTS project directly to the choroidal subdivision of the SSN. Following retrograde tracer injection into SSN (biotinylated dextran amine 3K or Fluorogold), labeled perikarya were found in PVN and NTS. Injection of the anterograde tracer, biotinylated dextran amine 10K (BDA10K), into PVN or NTS resulted in densely packed BDA10K+terminals in prechoroidal SSN (as defined by its enrichment in nitric oxide synthase-containing perikarya). Double-label studies showed these inputs ended directly on prechoroidal nitric oxide synthase-containing neurons of SSN. Our study thus establishes that PVN and NTS project directly to the part of SSN involved in parasympathetic vasodilatory control of the choroid via the PPG. These results suggest that control of ChBF may be linked to systemic blood pressure and central control of the systemic vasculature.

Cerebellar cortical output encodes temporal aspects of rhythmic licking movements and is necessary for normal licking frequency.

Rodents consume water by performing stereotypic, rhythmic licking movements that are believed to be controlled by brainstem pattern-generating circuits. Previous work has shown that synchronized population activity of inferior olive neurons was phase-locked to the licking rhythm in rats, suggesting a cerebellar involvement in temporal aspects of licking behavior. However, what role the cerebellum has in licking behavior and whether licking is represented in the high-frequency simple spike output of Purkinje cells remains unknown. We recorded Purkinje cell simple and complex spike activity in awake mice during licking, and determined the behavioral consequences of loss of cerebellar function. Mouse cerebellar cortex contained a multifaceted representation of licking behavior encoded in the simple spike activities of Purkinje cells distributed across Crus I, Crus II and lobus simplex of the right cerebellar hemisphere. Lick-related Purkinje cell simple spike activity was modulated rhythmically, phase-locked to the lick rhythm, or non-rhythmically. A subpopulation of lick-related Purkinje cells differentially represented lick interval duration in their simple spike activity. Surgical removal of the cerebellum or temporary pharmacological inactivation of the cerebellar nuclei significantly slowed the licking frequency. Fluid licking was also less efficient in mice with impaired cerebellar function, indicated by a significant decline in the volume per lick fluid intake. The gross licking movement appeared unaffected. Our results suggest a cerebellar role in modulating the frequency of the central pattern-generating circuits controlling fluid licking and in the fine coordination of licking, while contributing little to the coordination of the gross licking movement.

High-throughput mutational analysis of TOR1A in primary dystonia.

BACKGROUND: Although the c.904_906delGAG mutation in Exon 5 of TOR1A typically manifests as early-onset generalized dystonia, DYT1 dystonia is genetically and clinically heterogeneous. Recently, another Exon 5 mutation (c.863G>A) has been associated with early-onset generalized dystonia and some DeltaGAG mutation carriers present with late-onset focal dystonia. The aim of this study was to identify TOR1A Exon 5 mutations in a large cohort of subjects with mainly non-generalized primary dystonia. METHODS: High resolution melting (HRM) was used to examine the entire TOR1A Exon 5 coding sequence in 1014 subjects with primary dystonia (422 spasmodic dysphonia, 285 cervical dystonia, 67 blepharospasm, 41 writer's cramp, 16 oromandibular dystonia, 38 other primary focal dystonia, 112 segmental dystonia, 16 multifocal dystonia, and 17 generalized dystonia) and 250 controls (150 neurologically normal and 100 with other movement disorders). Diagnostic sensitivity and specificity were evaluated in an additional 8 subjects with known DeltaGAG DYT1 dystonia and 88 subjects with DeltaGAG-negative dystonia. RESULTS: HRM of TOR1A Exon 5 showed high (100%) diagnostic sensitivity and specificity. HRM was rapid and economical. HRM reliably differentiated the TOR1A DeltaGAG and c.863G>A mutations. Melting curves were normal in 250/250 controls and 1012/1014 subjects with primary dystonia. The two subjects with shifted melting curves were found to harbor the classic DeltaGAG deletion: 1) a non-Jewish Caucasian female with childhood-onset multifocal dystonia and 2) an Ashkenazi Jewish female with adolescent-onset spasmodic dysphonia. CONCLUSION: First, HRM is an inexpensive, diagnostically sensitive and specific, high-throughput method for mutation discovery. Second, Exon 5 mutations in TOR1A are rarely associated with non-generalized primary dystonia.

Cerebral cortical control of orbicularis oculi motoneurons.

Cerebral cortical neural networks associated with eyelid movement play a critical role in facial animation, contribute to the regulation of blink frequency, and help prevent ocular injury. Eyelid closure depends, in part, on motoneurons that innervate the orbicularis oculi (OO) muscles. In this study, OO motoneuron cortical afferents were identified in rhesus monkeys with rabies virus, a retrograde transneuronal tracer. Virus was injected into the right OO muscle and immunohistochemically localized after 4-6 day transport intervals. Labeled motoneurons were limited to dorsal portions of the ipsilateral facial motor nucleus. After 4- and 4.5-day transport intervals, most labeled cortical neurons were localized to ventrolateral premotor (LPMCv), dorsolateral premotor (LPMCd), and motor (M1) cortices. Labeled neurons were more sparsely distributed in supplementary (M2), caudal (M4), and rostral (M3) cingulate motor cortices; the frontal eye fields (FEF); pre-supplementary motor cortex (pre-SMA); somatosensory cortices (areas 3a, 3b, and 1); and prefrontal cortex. At longer transport intervals (5-6 days), labeled neurons increased substantially in LPMCv, LPMCd, M2, M3, M4, pre-SMA, and FEF. Concentrations of labeled neurons also appeared in cortices along the lateral fissure and intraparietal sulcus. Overall, the densest collection of labeled neurons was localized to the caudal junction of LPMCd and LPMCv with M1. Rostral M3 was another focus of OO premotor neurons. Labeled neurons were distributed bilaterally in all motor cortical areas with a modest contralateral predominance for M2, LPMC, and M1. Thus, the cortical control of OO motor activity is distributed bilaterally among multiple motor areas.

Developmental expression of rat torsinA transcript and protein.

A GAG deletion in the gene (TOR1A) for torsinA is associated with childhood-onset generalized dystonia (DYT1). Environmental factors may contribute to development of the phenotype since mutations in TOR1A are clinically penetrant in less than 40% of cases. Median age of onset is 10 and appearance of dystonia after 28 is rare. As a step towards understanding the temporal window of DYT1 disease penetrance, we have examined torsinA transcript and protein expression in rats from the embryonic period through adulthood. With relative quantitative multiplex real-time RT-PCR, we detected torsinA transcript in both neural (cerebellar cortex, striatum, cerebral cortex, thalamus and hippocampus) and non-neural (liver, kidney and heart) tissues at each developmental time point tested (embryonic day 20 [E20], postnatal day 1 [P1], P7, P14, P36, 6 months, 1.5 years). Levels of torsinA transcript were highest at E20 or P1 in all tissues examined except for the cerebellum where transcript levels peaked at P14. Early postnatal levels of torsinA transcript were over three times higher than those seen in adult rats. With quantitative radioactive in situ hybridization, torsinA transcript was widely distributed in brain at all ages with levels peaking at P14 in both cerebellum and striatum. TorsinA-immunoreactivity (IR) was present in neurons throughout the brain. TorsinA-IR was detected in perikarya, dendrites and axons but not nuclei. At P14, prominent expression of torsinA was noted in both striatal cholinergic interneurons and cerebellar Purkinje cells. Our results suggest that torsinA may contribute to postnatal maturational events in the brain such as dendritic arborization and synaptogenesis. Furthermore, the time course of torsinA expression in discrete components of motor networks is compatible with the temporal window of clinical penetrance in DYT1 mutation carriers.

Blink-related sensorimotor anatomy in the rat.

Protection of the eye and maintenance of the precorneal tear film depend on sensory innervation of the cornea and eyelids and motor innervation of muscles involved in closing and opening the eyes. Using a variety of fluorescent and transganglionic tracers, the sensorimotor innervation of blink-related orbital and periorbital structures was studied in Sprague-Dawley rats. The orbicularis oculi muscle surrounded the entire palpebral fissure and was innervated by motoneurons located along the dorsal cap of the ipsilateral facial motor nucleus. Upper and lower eyelid orbicularis oculi motoneurons were strictly ipsilateral and co-extensive, but upper eyelid orbicularis oculi motoneurons were, on average, slightly rostral and lateral to lower eyelid orbicularis oculi motoneurons. Facial motoneurons supplying the frontoscutularis, a muscle that helps to elevate the upper eyelid, were located in the medial division of the ipsilateral facial motor nucleus. Presumptive type Abeta afferents from the cornea terminated most prominently at the junction of the first cervical segment and the spinal trigeminal nucleus, pars caudalis. There was a second concentration of corneal terminations at the junction of pars caudalis and pars interpolaris of the spinal trigeminal nucleus. Sparse projections to the spinal trigeminal nucleus, pars oralis and the principal trigeminal nucleus were also detected. Presumptive type Abeta afferents from the eyelids terminated throughout the rostrocaudal extent of the spinal trigeminal nucleus with a heavy concentration within laminae III and IV of the first cervical segment. Presumptive types Adelta and C terminals from the eyelids were virtually limited to laminae I and II of the first cervical segment. Central terminations from the frontal nerve were present in the principal trigeminal nucleus and throughout the spinal trigeminal nucleus, but were most prominent within the dorsal horn of the first cervical segment. Our comprehensive description of blink-related sensorimotor anatomy in rats will provide a foundation for future physiological studies of blinking.

Localization of preganglionic neurons that innervate choroidal neurons of pterygopalatine ganglion.

PURPOSE: The pterygopalatine ganglion (PPG) receives preganglionic input from the superior salivatory nucleus (SSN) of the facial motor complex and is the main source of parasympathetic input to the choroid in mammals. The present study was undertaken to determine in rats the location and neurotransmitters of SSN neurons innervating those PPG neurons that target the choroid and to determine the location and neurotransmitters of the PPG choroidal neurons themselves. METHODS: Retrograde labeling from rat choroid using a fluorescent tracer, in combination with immunofluorescence labeling for nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), and choline acetyltransferase (ChAT), was used to characterize the location and neurotransmitters of choroidal PPG neurons. To identify SSN neurons that innervate the choroidal PPG neurons, the Bartha strain of the retrograde transneuronal tracer pseudorabies virus (PRV-Ba) was injected into rat choroid, and immunolabeling for NOS or ChAT was used to characterize their neurochemistry. RESULTS: Fluorescent retrograde labeling showed that PPG neurons projecting to the choroid contained NOS, VIP, and ChAT and were widely distributed in PPG and its preganglionic root, the greater petrosal nerve. SSN neurons were ChAT(+), and a subset of them was found to contain NOS. PRV-Ba transneuronal retrograde labeling revealed that choroidal preganglionic neurons were localized to the rostral medioventral part of the ipsilateral SSN. The choroidal SSN neurons were ChAT(+) and appeared largely to correspond to the NOS(+) neurons of the SSN. CONCLUSIONS: These results show that preganglionic neurons in rats that are presumed to regulate choroidal blood flow through the PPG reside within the rostral medioventral SSN, and that NOS is a marker for these SSN neurons.

Immunohistochemical detection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP).

Traditional histochemical detection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) can impose substantial technical limitations on studies requiring co-localization of neurotransmitters, receptors and other neural antigens. The goal of our experiments was to establish the ideal conditions and reagents for immunohistochemical detection of WGA-HRP. WGA-HRP was injected into the tongues and vibrissae pads of adult rats to characterize labeling of somas and synapses, respectively. Rats were perfused with either 4% paraformaldehyde (for light microscopy, LM) or 4% paraformaldehyde/0.15% glutaraldehyde (for electron microscopy, EM) after survival times of 2, 3, 4, 5 or 6 days. For LM, brainstem tissue was cut on a cryostat at 20 microm and collected onto glass slides. For EM, tissue was sectioned with a vibratome at 50 microm and processed free floating. For LM, WGA-HRP was detected with goat anti-HRP, goat anti-WGA, biotinylated goat anti-HRP or biotinylated goat anti-WGA antibodies. For EM, WGA-HRP was detected with biotinylated goat anti-WGA and anti-HRP antibodies. Survival intervals of 3 days were ideal for staining of hypoglossal neurons, whereas an interval of 4 days produced the strongest staining of synapses within the spinal trigeminal nucleus. For LM, the biotinylated antibodies resulted in better signal-to-noise ratios than the unconjugated antibodies. At both the LM and EM levels, the biotinylated antibody to WGA produced better quality staining than the biotinylated antibody to HRP.