Coordinated Postnatal Maturation of Striatal Cholinergic Interneurons and Dopamine Release Dynamics in Mice.

Dynamic changes in motor abilities and motivated behaviors occur during the juvenile and adolescent periods. The striatum is a subcortical nucleus critical to action selection, motor learning, and reward processing. Its tonically active cholinergic interneuron (ChI) is an integral regulator of the synaptic activity of other striatal neurons, as well as afferent axonal projections of midbrain dopamine (DA) neurons; however, little is known about its development. Here, we report that ChI spontaneous activity increases during postnatal development of male and female mice, concomitant with a decreased afterhyperpolarization (AHP). We characterized the postnatal development of four currents that contribute to the spontaneous firing rate of ChIs, including ISK, IA, Ih, and INaP We demonstrated that the developmental increase in INaP drives increased ChI firing rates during the postnatal period and can be reversed by the INaP inhibitor, ranolazine. We next addressed whether immature cholinergic signaling may lead to functional differences in DA release during the juvenile period. In the adult striatum, nicotinic acetylcholine receptors (nAChRs) prevent linear summation of DA release in response to trains of high-frequency stimuli. We show that, in contrast, during the second postnatal week, DA release linearly sums with trains of high-frequency stimuli. Consistently, nAChR antagonists exert little effect on dopamine release at postnatal day (P)10, but enhance the summation of evoked DA release in mice older than postnatal day P28. Together, these results reveal that postnatal maturation of ChI activity is due primarily to enhanced INaP and identify an interaction between developing cholinergic signaling and DA neurotransmission in the juvenile striatum.SIGNIFICANCE STATEMENT Motor skills and motivated behavior develop rapidly in juvenile rodents. Recent work has highlighted processes that contribute to the postnatal maturation of striatal principal neurons during development. The functional development of the striatal cholinergic interneuron (ChI), however, has been unexplored. In this study, we tracked the ontogeny of ChI activity and cellular morphology, as well as the developmental trajectory of specific conductances that contribute to the activity of these cells. We further report a link between cholinergic signaling and dopamine (DA) release, revealing a change in the frequency-dependence of DA release during the early postnatal period that is mediated by cholinergic signaling. This study provides evidence that striatal microcircuits are dynamic during the postnatal period and that they undergo coordinated maturation.

Cancer-Associated Neurogenesis and Nerve-Cancer Cross-talk.

In this review, we highlight recent discoveries regarding mechanisms contributing to nerve-cancer cross-talk and the effects of nerve-cancer cross-talk on tumor progression and dissemination. High intratumoral nerve density correlates with poor prognosis and high recurrence across multiple solid tumor types. Recent research has shown that cancer cells express neurotrophic markers such as nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor and release axon-guidance molecules such as ephrin B1 to promote axonogenesis. Tumor cells recruit new neural progenitors to the tumor milieu and facilitate their maturation into adrenergic infiltrating nerves. Tumors also rewire established nerves to adrenergic phenotypes via exosome-induced neural reprogramming by p53-deficient tumors. In turn, infiltrating sympathetic nerves facilitate cancer progression. Intratumoral adrenergic nerves release noradrenaline to stimulate angiogenesis via VEGF signaling and enhance the rate of tumor growth. Intratumoral parasympathetic nerves may have a dichotomous role in cancer progression and may induce Wnt-ß-catenin signals that expand cancer stem cells. Importantly, infiltrating nerves not only influence the tumor cells themselves but also impact other cells of the tumor stroma. This leads to enhanced sympathetic signaling and glucocorticoid production, which influences neutrophil and macrophage differentiation, lymphocyte phenotype, and potentially lymphocyte function. Although much remains unexplored within this field, fundamental discoveries underscore the importance of nerve-cancer cross-talk to tumor progression and may provide the foundation for developing effective targets for the inhibition of tumor-induced neurogenesis and tumor progression.

Autonomic nervous system functioning in early childhood: Responses to cognitive and negatively valenced emotional challenges.

Although autonomic nervous system (ANS) functioning is "context-dependent," few studies examined children's normative sympathetic and parasympathetic autonomic responses to distinct challenges in early childhood years. Examining children's ANS responsivity to distinct challenges is important for understanding normative autonomic responses toward everyday life stressors and identifying paradigms that effectively elicit a "stress response." We examined children's (N = 278) sympathetic (preejection period [PEP]) and parasympathetic (respiratory sinus arrhythmia [RSA]) responses to cognitive (i.e., problem-solving and cognitive control) and negatively valenced emotional (i.e., blocked goal and unfairness) challenges in preschool, kindergarten, and grade 1. Children, on average, demonstrated parasympathetic inhibition (RSA withdrawal) in response to all challenges but the magnitude of these responses depended on the task. Children showed sympathetic activation (PEP shortening) toward the problem-solving task at each assessment and there was no sample-level change in the magnitude of this response over time. Children showed greater sympathetic responsivity toward the cognitive control task over time, with evidence for a sympathetic activation response only in grade 1. Children experienced sympathetic inhibition (PEP lengthening) toward the unfairness tasks but did not experience significant sympathetic responsivity toward the blocked goal tasks. Parasympathetic responsivity to most challenges were modestly stable but there was no stability in sympathetic responsivity across time.

Features of the structure, development, and activity of the zebrafish noradrenergic system explored in new CRISPR transgenic lines.

The noradrenergic (NA) system of vertebrates is implicated in learning, memory, arousal, and neuroinflammatory responses, but is difficult to access experimentally. Small and optically transparent, larval zebrafish offer the prospect of exploration of NA structure and function in an intact animal. We made multiple transgenic zebrafish lines using the CRISPR/Cas9 system to insert fluorescent reporters upstream of slc6a2, the norepinephrine transporter gene. These lines faithfully express reporters in NA cell populations, including the locus coeruleus (LC), which contains only about 14 total neurons. We used the lines in combination with two-photon microscopy to explore the structure and projections of the NA system in the context of the columnar organization of cell types in the zebrafish hindbrain. We found robust alignment of NA projections with glutamatergic neurotransmitter stripes in some hindbrain segments, suggesting orderly relations to neuronal cell types early in life. We also quantified neurite density in the rostral spinal cord in individual larvae with as much as 100% difference in the number of LC neurons, and found no correlation between neuronal number in the LC and projection density in the rostral spinal cord. Finally, using light sheet microscopy, we performed bilateral calcium imaging of the entire LC. We found that large-amplitude calcium responses were evident in all LC neurons and showed bilateral synchrony, whereas small-amplitude events were more likely to show interhemispheric asynchrony, supporting the potential for targeted LC neuromodulation. Our observations and new transgenic lines set the stage for a deeper understanding of the NA system.

Otitis media and respiratory sinus arrhythmia across infancy and early childhood: Polyvagal processes?

Otitis media (OM)-or middle-ear inflammation-is the most widely diagnosed childhood illness, with evidence implicating OM in a range of distal problems (e.g., language delays, attention problems). Polyvagal theory (Porges, 1995, 2007) posits that there also are likely important connections between middle-ear functioning and children's developing parasympathetic nervous systems (PNS). Using prospective longitudinal data from the Family Life Project (n = 748), we tested within- and between-person relations between indicators of OM (middle-ear spectral gradient angle; SGA) and children's trajectories of respiratory sinus arrhythmia (RSA)-a marker of parasympathetic control of the heart-between the ages of 7 and 35 months. The results suggested that, irrespective of age, children with indications of chronic OM (low cumulative SGA) tended to show atypical RSA reactivity to moderate cognitive challenge, compared with the reactivity patterns of their low-OM-risk peers (mid-to-high cumulative SGA). Specifically, on average, low-OM-risk children showed RSA decreases in the context of challenge in infancy, with the magnitude of the decline weakening and eventually changing direction (i.e., RSA increase) by 35 months. In contrast, those with indicators of chronic OM evinced blunted RSA responses to challenge, irrespective of age. Within-person, temporal bouts of OM-risk were not predictive of within-person changes in RSA reactivity across early childhood. (PsycINFO Database Record

Cholinergic innervation of principal neurons in the cochlear nucleus of the Mongolian gerbil.

Principal neurons in the ventral cochlear nucleus (VCN) receive powerful ascending excitation and pass on the auditory information with exquisite temporal fidelity. Despite being dominated by ascending inputs, the VCN also receives descending cholinergic connections from olivocochlear neurons and from higher regions in the pontomesencephalic tegmentum. In Mongolian gerbils, acetylcholine acts as an excitatory and modulatory neurotransmitter on VCN neurons, but the anatomical structure of cholinergic innervation of gerbil VCN is not well described. We applied fluorescent immunohistochemical staining to elucidate the development and the cellular localization of presynaptic and postsynaptic components of the cholinergic system in the VCN of the Mongolian gerbil. We found that cholinergic fibers (stained with antibodies against the vesicular acetylcholine transporter) were present before hearing onset at P5, but innervation density increased in animals after P10. Early in development cholinergic fibers invaded the VCN from the medial side, spread along the perimeter and finally innervated all parts of the nucleus only after the onset of hearing. Cholinergic fibers ran in a rostro-caudal direction within the nucleus and formed en-passant swellings in the neuropil between principal neurons. Nicotinic and muscarinic receptors were expressed differentially in the VCN, with nicotinic receptors being mostly expressed in dendritic areas while muscarinic receptors were located predominantly in somatic membranes. These anatomical data support physiological indications that cholinergic innervation plays a role in modulating information processing in the cochlear nucleus.

Similar synapse elimination motifs at successive relays in the same efferent pathway during development in mice.

In many parts of the nervous system, signals pass across multiple synaptic relays on their way to a destination, but little is known about how these relays form and the function they serve. To get some insight into this question we ask how the connectivity patterns are organized at two successive synaptic relays in a simple, cholinergic efferent pathway. We found that the organization at successive relays in the parasympathetic nervous system strongly resemble each other despite the different embryological origin and physiological properties of the pre- and postsynaptic cells. Additionally, we found a similar developmental synaptic pruning and elaboration strategy is used at both sites to generate their adult organizations. The striking parallels in adult innervation and developmental mechanisms at the relays argue that a general strategy is in operation. We discuss why from a functional standpoint this structural organization may amplify central signals while at the same time maintaining positional targeting.

Autonomic nerve development contributes to prostate cancer progression.

Nerves are a common feature of the microenvironment, but their role in tumor growth and progression remains unclear. We found that the formation of autonomic nerve fibers in the prostate gland regulates prostate cancer development and dissemination in mouse models. The early phases of tumor development were prevented by chemical or surgical sympathectomy and by genetic deletion of stromal ß2- and ß3-adrenergic receptors. Tumors were also infiltrated by parasympathetic cholinergic fibers that promoted cancer dissemination. Cholinergic-induced tumor invasion and metastasis were inhibited by pharmacological blockade or genetic disruption of the stromal type 1 muscarinic receptor, leading to improved survival of the mice. A retrospective blinded analysis of prostate adenocarcinoma specimens from 43 patients revealed that the densities of sympathetic and parasympathetic nerve fibers in tumor and surrounding normal tissue, respectively, were associated with poor clinical outcomes. These findings may lead to novel therapeutic approaches for prostate cancer.

Developmental changes in GABAergic neurotransmission to presympathetic and cardiac parasympathetic neurons in the brainstem.

Cardiovascular function is regulated by a dynamic balance composed of sympathetic and parasympathetic activity. Sympathoexcitatory presympathetic neurons (PSNs) in the rostral ventrolateral medulla project directly to cardiac and vasomotor sympathetic preganglionic neurons in the spinal cord. In proximity to the PSNs in the medulla, there are preganglionic cardiac vagal neurons (CVNs) within the nucleus ambiguus, which are critical for parasympathetic control of heart rate. Both CVNs and PSNs receive GABAergic synaptic inputs that change with challenges such as hypoxia and hypercapnia (H/H). Autonomic control of cardiovascular function undergoes significant changes during early postnatal development; however, little is known regarding postnatal maturation of GABAergic neurotransmission to these neurons. In this study, we compared changes in GABAergic inhibitory postsynaptic currents (IPSCs) in CVNs and PSNs under control conditions and during H/H in postnatal day 2-5 (P5), 16-20 (P20), and 27-30 (P30) rats using an in vitro brainstem slice preparation. There was a significant enhancement in GABAergic neurotransmission to both CVNs and PSNs at age P20 compared with P5 and P30, with a more pronounced increase in PSNs. H/H did not significantly alter this enhanced GABAergic neurotransmission to PSNs in P20 animals. However, the frequency of GABAergic IPSCs in PSNs was reduced by H/H in P5 and P30 animals. In CVNs, H/H elicited an inhibition of GABAergic neurotransmission in all ages studied, with the most pronounced inhibition occurring at P20. In conclusion, there are critical development periods at which significant rearrangement occurs in the central regulation of cardiovascular function.

Neonatal leptin exposure specifies innervation of presympathetic hypothalamic neurons and improves the metabolic status of leptin-deficient mice.

The paraventricular nucleus of the hypothalamus (PVH) consists of distinct functional compartments regulating neuroendocrine, behavioral, and autonomic activities that are involved in the homeostatic control of energy balance. These compartments receive synaptic inputs from neurons of the arcuate nucleus of the hypothalamus (ARH) that contains orexigenic agouti-related peptide (AgRP) and anorexigenic pro-opiomelanocortin (POMC) neuropeptides. The axon outgrowth from the ARH to PVH occurs during a critical postnatal period and is influenced by the adipocyte-derived hormone leptin, which promotes its development. However, little is known about leptin's role in specifying patterns of cellular connectivity in the different compartments of the PVH. To address this question, we used retrograde and immunohistochemical labeling to evaluate neuronal inputs onto sympathetic preautonomic and neuroendocrine neurons in PVH of leptin-deficient mice (Lep(ob)/Lep(ob)) exposed to a postnatal leptin treatment. In adult Lep(ob)/Lep(ob) mice, densities of AgRP- and α-melanocortin stimulating hormone (αMSH)-immunoreactive fibers were significantly reduced in neuroendocrine compartments of the PVH, but only AgRP were reduced in all regions containing preautonomic neurons. Moreover, postnatal leptin treatment significantly increased the density of AgRP-containing fibers and peptidergic inputs onto identified preautonomic, but not onto neuroendocrine cells. Neonatal leptin treatment neither rescued αMSH inputs onto neuroendocrine neurons, nor altered cellular ratios of inhibitory and excitatory inputs. These effects were associated with attenuated body weight gain, food intake and improved physiological response to sympathetic stimuli. Together, these results provide evidence that leptin directs cell type-specific patterns of ARH peptidergic inputs onto preautonomic neurons in the PVH, which contribute to normal energy balance regulation.

The sox gene Dichaete is expressed in local interneurons and functions in development of the Drosophila adult olfactory circuit.

In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. We have found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete-expressing local interneurons in development of the adult olfactory circuitry.

Relationships between QT interval and heart rate variability at rest and the covariates in healthy young adults.

To clarify the links between ECG QT-related parameters and heart rate variability (HRV) and the covariates possibly distorting them, the averaged RR and QT intervals in a single lead ECG were measured for 64 male and 86 female subjects aged 18-26. The QT index, defined by Rautaharju et al., in the young adults was not significantly related to any HRV parameters nor heart rate, but the Bazett's corrected QT (QTc) interval was associated negatively with the parasympathetic activity and positively with heart rate. No significant differences in the QTc interval, QT index or heart rate were seen between the men and women, but they significantly differed between both sexes after adjustment for possible covariates such as age and body mass index (BMI). Significant sex differences in parasympathetic parameters of the HRV were unchanged before and after the adjustment, but significant differences observed in the unadjusted sympathetic parameters disappeared after adjusting for covariates. Age, BMI and body fat percentage also were significant covariates affecting these ECG parameters. Consequently, QT index, unaffected by heart rate and HRV parameters, appears to be a more useful indicator than the QTc interval. Instead, the QT index and HRV parameters are recommended to be simultaneously measured in epidemiological research because they are probably complementary in assessing autonomic nervous function. Also, these parameters should be analyzed in men and women separately.

Detailed comparative anatomy of the extrinsic cardiac nerve plexus and postnatal reorganization of the cardiac position and innervation in the great apes: orangutans, gorillas, and chimpanzees.

To speculate how the extrinsic cardiac nerve plexus (ECNP) evolves phyletically and ontogenetically within the primate lineage, we conducted a comparative anatomical study of the ECNP, including an imaging examination in the great apes using 20 sides from 11 bodies from three species and a range of postnatal stages from newborns to mature adults. Although the position of the middle cervical ganglion (MG) in the great apes tended to be relatively lower than that in humans, the morphology of the ECNP in adult great apes was almost consistent with that in adult humans but essentially different from that in the lesser apes or gibbons. Therefore, the well-argued anatomical question of when did the MG acquire communicating branches with the spinal cervical nerves and appear constantly in all sympathetic cardiac nerves during primate evolution is clearly considered to be after the great apes and gibbons split. Moreover, a horizontal four-chambered heart and a lifted cardiac apex with a relatively large volume in newborn great apes rapidly changed its position downward, as seen in humans during postnatal growth and was associated with a reduction in the hepatic volume by imaging diagnosis and gross anatomy. In addition, our observation using a range of postnatal stages exhibits that two sympathetic ganglia, the middle cervical and cervicothoracic ganglia, differed between the early and later postnatal stages.

Nicotine exposure during the third trimester equivalent of human gestation: time course of effects on the central cholinergic system of rats.

Up to 22% of pregnant women smoke, which constitutes a major health concern. Nicotine, a cholinergic agonist, causes deleterious effects on brain development. However, most studies investigate its effects during rodents' gestation, which corresponds, in terms of neural development, to the first two trimesters of human gestation. Here, we focused on effects of nicotine on the brain cholinergic system during the third trimester equivalent of human gestation. From the 2nd to the 19th day of lactation, dams were exposed either to nicotine (6 mg/kg/day) or to saline via sc osmotic minipumps. Offspring were sacrificed during exposure (PN15, PN, postnatal) or at 2 days (PN21), 11 days (PN30), or 10 weeks (PN90) of withdrawal. In the cerebral cortex, midbrain, and hippocampus, we assessed nicotinic acetylcholine receptor (nAChR) binding, [(3)H]hemicholinium-3 (HC-3) binding to the high-affinity choline transporter, choline acetyltransferase (ChAT), and acetylcholinesterase (AChE) activities. Nicotine-exposed offspring presented nAChR upregulation during exposure in all brain regions, reduced HC-3 binding during and 11 days postexposure, and increased HC-3 binding on PN90. Effects on ChAT and AChE were dependent on the brain region and restricted to the withdrawal period: There were increased activities in the midbrain on PN30. In the hippocampus, AChE as reduced on PN30, whereas, for ChAT, the decrease was followed by late-emergent increased activity. These data indicate that maternal nicotine exposure during the third trimester equivalent of human gestation promotes cholinergic system alterations in the offspring's brain. In addition, detrimental effects are observable even long after the exposure has been interrupted.

Cardiovascular regulation profile predicts developmental trajectory of BMI and pediatric obesity.

The present study examined the role of cardiovascular regulation in predicting pediatric obesity. Participants for this study included 268 children (141 girls) obtained from a larger ongoing longitudinal study. To assess cardiac vagal regulation, resting measures of respiratory sinus arrhythmia (RSA) and RSA change (vagal withdrawal) to three cognitively challenging tasks were derived when children were 5.5 years of age. Heart period (HP) and HP change (heart rate (HR) acceleration) were also examined. Height and weight measures were collected when children were 5.5, 7.5, and 10.5 years of age. Results indicated that physiological regulation at age 5.5 was predictive of both normal variations in BMI development and pediatric obesity at age 10.5. Specifically, children with a cardiovascular regulation profile characterized by lower levels of RSA suppression and HP change experienced significantly greater levels of BMI growth and were more likely to be classified as overweight/at-risk for overweight at age 10.5 compared to children with a cardiovascular regulation profile characterized by high levels of RSA suppression and HP change. However, a significant interaction with racial status was found suggesting that the association between cardiovascular regulation profile and BMI growth and pediatric obesity was only significant for African-American children. An autonomic cardiovascular regulation profile consisting of low parasympathetic activity represents a significant individual risk factor for the development of pediatric obesity, but only for African-American children. Mechanisms by which early physiological regulation difficulties may contribute to the development of pediatric obesity are discussed.

Regulation of cardiac innervation and function via the p75 neurotrophin receptor.

Homeostatic regulation of cardiac function is dependent on the balance of inputs from the sympathetic and parasympathetic nervous systems. We investigated whether the p75 neurotrophin receptor plays a developmental role in cardiac innervation by analyzing sympathetic and parasympathetic fibers in the atria of p75 knockout and wildtype mice at several stages of postnatal development, and examining the effect on control of heart rate. We found that parasympathetic innervation of the atria in p75-/- mice was similar to wildtype at all time points, but that the density of sympathetic innervation was dynamically regulated. Compared to wildtype mice, the p75-/- mice had less innervation at postnatal day 4, an increase at day 28, and decreased innervation in adult mice. These changes reflect defects in initial fiber in-growth and the timing of the normal developmental decrease in sympathetic innervation density in the atria. Thus, p75 regulates both the growth and stability of cardiac sympathetic fibers. The distribution of sympathetic fibers was also altered, so that many regions lacked innervation. Basal heart rate was depressed in adult p75-/- mice, and these mice exhibited a diminished heart rate response to restraint stress. This resulted from the lack of sympathetic innervation rather than increased parasympathetic transmission or a direct effect of p75 in cardiac cells. Norepinephrine was elevated in p75-/- atria, but stimulating norepinephrine release with tyramine produced less tachycardia in p75-/- mice than wild type mice. This suggests that altered density and distribution of sympathetic fibers in p75-/- atria impairs the control of heart rate.

Autonomic nervous system activity in premature and full-term infants from theoretical term to 7 years.

The premature population reaching theoretical term suffers from a major deficit in autonomic nervous system (ANS) activity, as can be seen from heart rate variability indices. Whether this autonomic function recovers in the long term is not yet established. Thus, we analyzed and compared ANS activity indices, at birth or at the time of the theoretical term, and at ages 2-3 and 6-7 years, in two populations: a group of 30 premature children and a reference group of 14 full-term age-matched newborns. Using Fourier Transform analysis, we studied 24-h ECG Holter recordings to establish heart rate variability indices: Ptot, VLF, LF, HF, ratio LF/HF, LFnu, HFnu. In the neonatal period, sympathetic and even more markedly, parasympathetic activities were very low in prematures compared to the reference full-term group. At ages 2-3 and 6-7 years, prematures had recovered and had similar ANS activity as the full-term group. These data suggest a fast ANS maturation in prematures during the two first years of life, with a higher speed of recovery for the parasympathetic arm. Furthermore, compared evolution shows a faster ANS maturation in premature. Potential mechanisms are discussed.

Spontaneous awakening from nocturnal sleep and cardiac autonomic function in preschool children.

A cross-sectional study was carried out to clarify the physiological features of spontaneous awakening from nocturnal sleep (i.e., whether a child can spontaneously wake up on weekday mornings). The study population comprised 116 children at ages 5 and 6 years. Heart rate variability reflecting cardiac sympathetic and parasympathetic activities was measured. Children's typical bedtimes and wake times for weekdays and the presence/absence of spontaneous awakening from nocturnal sleep were reported by parents, and information about obligatory naptimes was provided by preschool teachers. The mean total sleep duration in the children was 625+/-56 (standard deviation) min. Total and nocturnal sleep durations were significantly shorter in 52 children without spontaneous awakening than in 64 children with it. Similarly, the parasympathetic activity was significantly lower in the children without spontaneous awakening, even in using analysis of covariance. Heart rate was significantly increased in the children without spontaneous awakening, but neither total nor nocturnal sleep durations were significant covariates in the analysis of covariance. In conclusion, the absence of spontaneous awakening from nocturnal sleep in preschool children is suggested to be characterized by short sleep duration, parasympathetic hypoactivity, and elevated heart rate.

Age-related changes in glycogen synthase kinase 3beta (GSK3beta) immunoreactivity in the central nervous system of rats.

Although glycogen synthase kinase 3beta (GSK3beta) is emerging as a prominent drug target in the treatment of neurodegenerative diseases such as Alzheimer's disease (AD) and stroke, very little is known about age-related changes in GSK3beta expression and GSK3beta phosphorylation. Therefore, we examined age-related changes in immunoreactivities for GSK3beta and phosphorylated GSK3beta (pGSK3beta) in the central nervous system. In aged rats, there were significant increases in GSK3beta immunoreactivity in the cell bodies and processes of pyramidal cells in most cortical regions. GSK3beta immunoreactivity was also significantly increased in the pyramidal layer of CA1-3 regions, and the granule cell layer of dentate gyrus. Age-related increases were prominent in lateral septal nuclei, compared to the medial septal nuclei. Interestingly, both GSK3beta and pGSK3beta was increased in the prefrontal cortex, while GSK3beta and pGSK3beta was differentially localized in the cerebellar cortex. The first demonstration of age-related alterations in immunoreactivities for GSK3beta and pGSK3beta in the basal forebrain area and cholinergic projection targets may provide useful data for investigating the pathogenesis of age-related neurodegenerative diseases including AD.

Effects of pituitary adenylate cyclase activating polypeptide on lumbosacral preganglionic neurons in the neonatal rat spinal cord.

The effects of PACAP-38 on phasic and tonic preganglionic neurons (PGN) in L6 and S1 spinal cord slices from neonatal rats (5--11 days old) were studied using the whole-cell patch clamp technique. PGN were identified by retrograde axonal transport of a fluorescent dye (Fast Blue, 5 microl of 4% solution) injected into the intraperitoneal space 3--7 days prior to the study. Bath application of pituitary adenylate cyclase activating polypeptide (PACAP) (20 nM) increased the frequency of spontaneous excitatory postsynaptic potentials (EPSPs) and spontaneous firing in both types of PGN. PACAP markedly increased the number (200--800%) and frequency of action potentials elicited by depolarizing current pulses in phasic PGN, but had a smaller effect on tonic PGN. PACAP decreased the threshold for action potential generation by approximately 25% in both types of neurons (e.g. -34.0+/-1.5 to -38.4+/-1.7 mV from a holding potential of -50 mV in phasic PGN, P<0.005). PACAP did not affect the duration of the action potential. The amplitude of the spike after hyperpolarization was not changed but the duration was significantly reduced by PACAP from 204.4+/-12.2 to 106.2+/-8.1 ms in tonic but not in phasic PGN. PACAP suppressed a transient outward current that was also suppressed by 4-aminopyridine (0.5 mM). These results coupled with the immunohistochemical identification of a dense collection of PACAP fibers in the region of the PGN, raises the possibility that PACAP may function as an excitatory transmitter in lumbosacral parasympathetic reflex pathways in the neonatal rat.