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.

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.

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.

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.

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.

Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory, and parasympathetic neurons.

Neurturin (NTN) is a neuronal survival factor that activates the Ret tyrosine kinase in the presence of a GPI-linked coreceptor (either GFR alpha1 or GFR alpha2). Neurturin-deficient (NTN-/-) mice generated by homologous recombination are viable and fertile but have defects in the enteric nervous system, including reduced myenteric plexus innervation density and reduced gastrointestinal motility. Parasympathetic innervation of the lacrimal and submandibular salivary gland is dramatically reduced in NTN-/- mice, indicating that Neurturin is a neurotrophic factor for parasympathetic neurons. GFR alpha2-expressing cells in the trigeminal and dorsal root ganglia are also depleted in NTN-/- mice. The loss of GFR alpha2-expressing neurons, in conjunction with earlier studies, provides strong support for GFR alpha2/Ret receptor complexes as the critical mediators of NTN function in vivo.

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.

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.

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.

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.

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.

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.

The GDNF family ligands and receptors - implications for neural development.

The glial cell line derived neurotrophic factor (GDNF) family has recently been expanded to include four members, and the interactions between these neurotrophic factors and their unique receptor system is now beginning to be understood. Furthermore, analysis of mice lacking the genes for GDNF, neurturin, and their related receptors has confirmed the importance of these factors in neurodevelopment. The results of such analyses reveal numerous similarities and potential overlaps in the way the GDNF and the nerve growth factor (NGF) families regulate development of the peripheral nervous system.

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.

Postnatal development of the putative neuropeptide-Y-mediated sympathetic--parasympathetic autonomic interaction.

OBJECTIVE: Intense stellate ganglion stimulation causes a long-lasting inhibition of cardiac vagal responses in adult dogs. This inhibition is thought to result from the release of neuropeptide Y from sympathetic nerve terminals, which, in turn, blocks the release of acetylcholine from parasympathetic neurons. The purpose of this study was to characterize the developmental expression of this autonomic interaction in the dog. METHODS: We studied and compared the effects of 5-min trains of right stellate ganglion stimulation on cardiac chronotropic responses to supramaximal vagal stimulation trains in 10 neonatal dogs, 8 one-month-old puppies, 8 two-month-old puppies and 8 adult dogs. RESULTS: In the adult group, after 5 min of stellate stimulation, inhibition of the vagal chronotropic response was observed in 7 of 8 (87.5%). Inhibition was observed in 100% of the one-month-olds and in 87.5% of the two-month-olds. In contrast, in the neonates, inhibition was observed in only 4 of 10 (40%) (P < 0.05). The maximum percent inhibition of the cardiac vagal response was significantly less in the neonates than in the older puppies (P < 0.001) and adults (P < 0.01), and the summated inhibition also tended to be less in the neonates (P < 0.05 compared to one- and two-month-old puppies). Finally, in 60% of the neonates and 37.5% of all other animals vagal responses after stellate stimulation were facilitated, i.e. at least 20% greater than the pre-stellate stimulation values. CONCLUSION: The putative neuropeptide-Y-mediated, sympathetic-parasympathetic interaction is not fully expressed in the canine neonate. It appears to develop quite rapidly postnatally, being fully expressed by 1 month of age. We hypothesize that this developmental change is likely the result of maturation of sympathetic nervous system function after birth. The facilitation of the vagal chronotropic response, observed in some animals after stellate stimulation, is a new finding, and may represent yet another type of autonomic interaction.

Development of the basal forebrain cholinergic system: phenotype expression prior to target innervation.

We measured choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in the rat to determine the time course of development, maturity, and senescence of ChAT activity. Tissue was obtained from Sprague-Dawley rats ranging in age from embryonic day 14 through 23 months. Seven regions were examined, including the magnocellular preoptic/substantia innominata region, frontal cortex, medial septal region, hippocampus, diagnoal band, and medial and lateral striatum. ChAT and AChE activities were first detected as early as E18 in the medial septum, diagonal band and magnocellular preoptic area, all regions of cholinergic cell bodies. Enzyme activity subsequently developed in terminal fields of these cholinergic perikarya (hippocampus and frontal cortex) as well as in the striatum. For all regions, enzyme activity rose during the first four postnatal weeks. This increase in enzyme activity was transient and, in most instances, decreases were observed between postnatal days 30 and 60. Most dramatic were the decreases in enzyme activity in the magnocellular preoptic/substantia innominata and diagonal band regions. Age-related declines also occurred in the frontal cortex, hippocampus, magnocellular preoptic/substantia innominata region, and the striatum. Cholinergic systems undergo dynamic changes especially during development and adulthood.

Dendritic maturation of displaced putative cholinergic amacrine cells in the rabbit retina.

The dendritic trees of Cb, cholinergic, amacrine cells in the ganglion cell layer of the developing rabbit retina are revealed by intracellular injection with Lucifer yellow to have the adult dendritic branching pattern at birth. It is demonstrated that these cells maintain a constant number of dendritic branches throughout postnatal development and that their dendritic trees increase in size by the growth and subsequent elongation of all branches. Proximal and distal dendrites increase in length by almost the same proportions between birth and adulthood. Although the adult pattern of dendritic branching of Cb amacrine cells is established by birth, dendrites in the young possess numerous short appendages (1-5 microns in length) resembling the "dendritic spines" of immature cat retinal ganglion cells. Some of these structures remain on the dendrites of adult cells but the majority are lost at the end of the third postnatal week. As dendritic spines disappear, the dendrites of Cb amacrine cells, especially the distal portion of the tree, acquire numerous varicosities. At each stage after P10, the gain in the number of varicosities greatly exceeds the loss in spines; this is not consistent with the hypothesis that all varicosities are retracted dendritic spines. The rapid increase in the number of varicosities on distal dendrites of Cb amacrine cells during the first 3 postnatal weeks coincides with the maturation of amacrine cell physiological responses. There is no distinct centroperipheral gradient in the postnatal dendritic maturation (acquisition of varicosities, loss of spines, attainment of the adult number of branches) of Cb amacrine cells from the visual streak to the peripheral retina. However, the area of their dendritic tree increases relatively more in the retinal periphery compared to that in the visual streak.

Postnatal addition of satellite cells to parasympathetic neurons.

We have examined the postnatal development of satellite cells associated with parasympathetic neurons of mouse salivary duct ganglia. The number of satellite cells associated with each neuron was found to increase during the first 8 weeks after birth but remained constant thereafter. This corresponds to the period of maximal growth of the salivary gland that serves as the target organ innervated by these neurons. At all ages examined, the number of satellite cells associated with each neuron was found to be highly correlated with neuronal volume. The development of satellite cells associated with individual identified neurons was followed directly by in vivo video microscopy over several months, and the number of satellite cell nuclei was found to increase in regions of the neuronal surface with increasing numbers of synaptic boutons. These results indicate that the postnatal addition of satellite cells to parasympathetic neurons is linked to neuronal enlargement and that synaptic remodeling occurs in concert with satellite cell development.

Pattern formation in the developing superior colliculus: ontogeny of the periodic architecture in the intermediate layers.

The superior colliculus of mammals contains a striking neurochemical architecture in which histochemically identifiable compartments are distributed in an iterative arrangement in the intermediate layers. We used stains for acetylcholinesterase activity as a compartment marker to trace ontogenesis of this architecture during pre- and postnatal development in the domestic cat. We found that compartmentation in the intermediate collicular layers is virtually absent at birth, and only gradually emerges during the first weeks of postnatal life. Over the same postnatal period, acetylcholinesterase activity shifts from a predominantly perikaryal expression pattern immediately postnatally to a nearly exclusive localization in the neuropil at maturity. Remarkably, a striking compartmentation of the superior colliculus was readily apparent with acetylcholinesterase histochemistry prenatally. The first appearance of a periodic architecture in the superior colliculus was observed at embryonic day 34, a time at which the collicular plate had not yet become laminated. The compartments characterized by high levels of acetylcholinesterase activity then gained in prominence until late in the prenatal period, when they receded and disappeared. The loss of the acetylcholinesterase-positive compartments in the perinatal period did not reflect a loss of compartmentation altogether. Neonatally, there was a distinct compartmental architecture visible with enkephalin immunohistochemistry. The virtual absence of acetylcholinesterase-positive compartments in the superior colliculus at birth therefore reflects developmental regulation of enzyme expression in the compartments, not regulation of the compartments as structural entities. We conclude that the periodic architecture, which characterizes the intermediate collicular layers in the adult cat, arises early in ontogenesis. These observations raise the possibility that the histochemical compartments are ontogenetic units that undergo remodeling as the superior colliculus matures.