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The cholinergic neurons in the pontomesencephalic tegmentum have been shown to discharge in association with and promote cortical activation during active or attentive waking and paradoxical or rapid eye movement sleep. However, GABA neurons lie intermingled with the cholinergic neurons and may contribute to or oppose this activity and role. Here we investigated in vitro and in vivo the properties, activities, and role of GABA neurons within the laterodorsal tegmental and sublaterodorsal tegmental nuclei (LDT/SubLDT) using male and female transgenic mice expressing channelrhodopsin-( ChR2 )- EYFP in vesicular GABA transporter ( VGAT )-expressing neurons. Presumed GABA (pGABA) neurons were identified by response to photostimulation and verified by immunohistochemical staining following juxtacellular labeling in vivo pGABA neurons were found to be fast-firing neurons with the capacity to burst when depolarized from a hyperpolarized membrane potential. When stimulated in vivo in urethane-anesthetized or unanesthetized mice, the pGABA neurons fired repetitively at relatively fast rates (∼40 Hz) during a continuous light pulse or phasically in bursts (>100 Hz) when driven by rhythmic light pulses at theta (4 or 8 Hz) frequencies. pNon-GABA, which likely included cholinergic, neurons were inhibited during each light pulse to discharge rhythmically in antiphase to the pGABA neurons. The reciprocal rhythmic bursting by the pGABA and pNon-GABA neurons drove rhythmic theta activity in the EEG. Such phasic bursting by GABA neurons also occurred in WT mice in association with theta activity during attentive waking and paradoxical sleep. SIGNIFICANCE STATEMENT Neurons in the pontomesencephalic tegmentum, particularly cholinergic neurons, play an important role in cortical activation, which occurs during active or attentive waking and paradoxical or rapid eye movement sleep. Yet the cholinergic neurons lie intermingled with GABA neurons, which could play a similar or opposing role. Optogenetic stimulation and recording of these GABA neurons in mice revealed that they can discharge in rhythmic bursts at theta frequencies and drive theta activity in limbic cortex. Such phasic burst firing also occurs during natural attentive waking and paradoxical sleep in association with theta activity and could serve to enhance sensory-motor processing and memory consolidation during these states., (Copyright © 2020 the authors.)

Micturition requires precise control of bladder and urethral sphincter via parasympathetic, sympathetic and somatic motoneurons. This involves a spino-bulbospinal control circuit incorporating Barrington's nucleus in the pons (Barr). Ponto-spinal glutamatergic neurons that express corticotrophin-releasing hormone (CRH) form one of the largest Barr cell populations. Barr CRH neurons can generate bladder contractions, but it is unknown whether they act as a simple switch or provide a high-fidelity pre-parasympathetic motor drive and whether their activation can actually trigger voids. Combined opto- and chemo-genetic manipulations along with multisite extracellular recordings in urethane anaesthetised CRH Cre mice show that Barr CRH neurons provide a probabilistic drive that generates co-ordinated voids or non-voiding contractions depending on the phase of the micturition cycle. CRH itself provides negative feedback regulation of this process. These findings inform a new inferential model of autonomous micturition and emphasise the importance of the state of the spinal gating circuit in the generation of voiding., Competing Interests: HI, AS, CF, AK, MD, AP No competing interests declared, (© 2020, Ito et al.)

Many species execute ballistic escape reactions to avoid imminent danger. Despite fast reaction times, responses are often highly regulated, reflecting a trade-off between costly motor actions and perceived threat level. However, how sensory cues are integrated within premotor escape circuits remains poorly understood. Here, we show that in zebrafish, less precipitous threats elicit a delayed escape, characterized by flexible trajectories, which are driven by a cluster of 38 prepontine neurons that are completely separate from the fast escape pathway. Whereas neurons that initiate rapid escapes receive direct auditory input and drive motor neurons, input and output pathways for delayed escapes are indirect, facilitating integration of cross-modal sensory information. These results show that rapid decision-making in the escape system is enabled by parallel pathways for ballistic responses and flexible delayed actions and defines a neuronal substrate for hierarchical choice in the vertebrate nervous system., Competing Interests: The authors have declared that no competing interests exist.

The basilar pontine nucleus (PN) is the key relay point for the cerebrocerebellar link. However, the projection pattern of pontocerebellar mossy fiber axons, which is essential in determining the functional organization of the cerebellar cortex, has not been fully clarified. We reconstructed the entire trajectory of 25 single pontocerebellar mossy fiber axons labeled by localized injection of biotinylated dextran amine into various locations in the PN and mapped all their terminals in an unfolded scheme of the cerebellum in 10 mice. The majority of axons (20/25 axons) entered the cerebellum through the middle cerebellar peduncle contralateral to the origin, while others entered through the ipsilateral pathway. A small number of axons (1/25 axons) had collaterals terminating in the cerebellar nuclei. Axons projected mostly to a combination of lobules, often bilaterally, and terminated in multiple zebrin (aldolase C) stripes, more frequently in zebrin-positive stripes (83.9%) than in zebrin-negative stripes, with 66.5 mossy fiber terminals on the average. Axons originating from the rostral (plus medial and lateral), central and caudal PN mainly terminated in the paraflocculus, crus I and lobule VIb-c, in the simplex lobule, crus II and paramedian lobule, and in lobules II-VIa, VIII and copula pyramidis, respectively. The results suggest that the interlobular branching pattern of pontocerebellar axons determines the group of cerebellar lobules that are involved in a related functional localization of the cerebellum. In the hemisphere, crus I may be functionally distinct from neighboring lobules (simple lobule and crus II) in the mouse cerebellum based on the pontocerebellar axonal projection pattern., (© 2019 Wiley Periodicals, Inc.)

The inner structure of the pons contains several layers of transverse and vertical fibers and many nuclei. The vertical bundles are described as fibers of the corticospinal tract, corticonuclear tract, frontopontine tract and parieto-temporopontine tract organized in three layers. The aim of this study was to investigate the structure of the vertical bundles in the ventral pons using the modified Klingler method. Ten brain stem specimens were investigated. Specimens were fixed in 10% formalin, frozen for 24 h to separate nerve fibers by ice crystals, and then unfrozen again in 10% formalin solution. Afterwards, the specimens were dissected using a sharpened spatula. Results point to the existence of three main layers of vertical bundles and a small, constant, and superficial fourth fascicle that is yet to be described in the literature. We propose the name fasciculus longitudinalis superficialis (superficial longitudinal fascicle) for this group of vertical fibers of the pons.

Sensory neurons of the jugular vagal ganglia innervate the respiratory tract and project to the poorly studied medullary paratrigeminal nucleus. In the present study, we used neuroanatomical tracing, pharmacology and physiology in guinea pig to investigate the paratrigeminal neural circuits mediating jugular ganglia-evoked respiratory reflexes. Retrogradely traced laryngeal jugular ganglia neurons were largely (> 60%) unmyelinated and expressed the neuropeptide substance P and calcitonin gene-related peptide, although a population (~ 30%) of larger diameter myelinated jugular neurons was defined by the expression of vGlut1. Within the brainstem, vagal afferent terminals were confined to the caudal two-thirds of the paratrigeminal nucleus. Electrical stimulation of the laryngeal mucosa evoked a vagally mediated respiratory slowing that was mimicked by laryngeal capsaicin application. These laryngeal reflexes were modestly reduced by neuropeptide receptor antagonist microinjections into the paratrigeminal nucleus, but abolished by ionotropic glutamate receptor antagonists. D,L-Homocysteic acid microinjections into the paratrigeminal nucleus mimicked the laryngeal-evoked respiratory slowing, whereas capsaicin microinjections evoked a persistent tachypnoea that was insensitive to glutamatergic inhibition but abolished by neuropeptide receptor antagonists. Extensive projections from paratrigeminal neurons were anterogradely traced throughout the pontomedullary respiratory column. Dual retrograde tracing from pontine and ventrolateral medullary termination sites, as well as immunohistochemical staining for calbindin and neurokinin 1 receptors, supported the existence of different subpopulations of paratrigeminal neurons. Collectively, these data provide anatomical and functional evidence for at least two types of post-synaptic paratrigeminal neurons involved in respiratory reflexes, highlighting an unrecognised complexity in sensory processing in this region of the brainstem.

Inhibitory afferent inputs to pontine A5 noradrenergic neurons (A5 NN) are not known, except partial baroreceptor input. In spontaneously breathing pentobarbital-anesthetized rats, we registered 35 A5 NN that were activated by hypoxia (100% N 2 , 10 sec) by more than 5 times in comparison with the background. Cooling of retrotrapezoid nucleus (15°C, 6 sec) completely blocked the motor inspiratory output and A5 NN discharge frequency increased (23/23) by more than 7 times in comparison with the background values. The beginning of A5 NN activation coincided with cessation of inspiratory activity. Short-term passive stretching of the shin muscles (1 sec, 100 g) caused BP drop and complete inhibition of A5 NN (12/12) activated by hypoxia. Inhibitory afferent inputs from proprioceptors and central inspiratory neurons that can limit A5 NN activity were demonstrated.

Key Points: Phasic activation of M1 muscarinic receptors generates transient inhibition followed by longer lasting excitation in neocortical pyramidal neurons. Corticopontine neurons in the mouse prefrontal cortex exhibit weaker cholinergic inhibition, but more robust and longer lasting excitation, than neighbouring callosal projection neurons. Optogenetic release of endogenous ACh in response to single flashes of light (5 ms) preferentially enhances the excitability of corticopontine neurons for many tens of seconds. Cholinergic excitation of corticopontine neurons involves at least three ionic mechanisms: suppression of K V 7 currents, activation of the calcium-dependent non-specific cation conductance underlying afterdepolarizations, and activation of what appears to be a calcium-sensitive but calcium-permeable non-specific cation conductance. Preferential cholinergic excitation of prefrontal corticopontine neurons may facilitate top-down attentional processes and behaviours., Abstract: Pyramidal neurons in layer 5 of the neocortex comprise two broad classes of projection neurons: corticofugal neurons, including corticopontine (CPn) neurons, and intratelencephalic neurons, including commissural/callosal (COM) neurons. These non-overlapping neuron subpopulations represent discrete cortical output channels contributing to perception, decision making and behaviour. CPn and COM neurons have distinct morphological and physiological characteristics, and divergent responses to modulatory transmitters such as serotonin and acetylcholine (ACh). To better understand how ACh regulates cortical output, in slices of mouse prefrontal cortex (PFC) we compared the responsivity of CPn and COM neurons to transient exposure to exogenous or endogenous ACh. In both neuron subtypes, exogenous ACh generated qualitatively similar biphasic responses in which brief hyperpolarization was followed by longer lasting enhancement of excitability. However, cholinergic inhibition was more pronounced in COM neurons, while excitatory responses were larger and longer lasting in CPn neurons. Similarly, optically triggered release of endogenous ACh from cholinergic terminals preferentially and persistently (for ∼40 s) enhanced the excitability of CPn neurons, but had little impact on COM neurons. Cholinergic excitation of CPn neurons involved at least three distinct ionic mechanisms: suppression of K V 7 channels (the 'M-current'), activation of the calcium-dependent non-specific cation conductance underlying afterdepolarizations, and activation of what appears to be a calcium-sensitive but calcium-permeable non-specific cation conductance. Our findings demonstrate projection-specific selectivity in cholinergic signalling in the PFC, and suggest that transient release of ACh during behaviour will preferentially promote corticofugal output., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

The A5 area at the ventrolateral pons contains noradrenergic neurons connected with other medullary areas involved in the cardiorespiratory control. Its contribution to the cardiorespiratory regulation was previously evidenced in anesthetized conditions. In the present study, we investigated the involvement of the A5 noradrenergic neurons to the basal and chemoreflex control of the sympathetic and respiratory activities in unanesthetized conditions. A5 noradrenergic neurons were lesioned using microinjections of anti-dopamine β-hydroxylase saporin (anti-DβH-SAP). After 7-8days, we evaluated the arterial pressure levels, heart rate and minute ventilation in freely moving adult rats (280-350g) as well as recorded from thoracic sympathetic (tSN) and phrenic nerves (PN) using the arterially perfused in situ preparation of juvenile rats (80-90g). Baseline cardiovascular, sympathetic and respiratory parameters were similar between control (n=7-8) and A5-lesioned rats (n=5-6) in both experimental preparations. In adult rats, lesions of A5 noradrenergic neurons did not modify the reflex cardiorespiratory adjustments to hypoxia (7% O 2 ) and hypercapnia (7% CO 2 ). In the in situ preparations, the sympatho-excitation, but not the PN reflex response, elicited by either the stimulation of peripheral chemoreceptors (ΔtSN: 110±12% vs 58±8%, P<0.01) or hypercapnia (ΔtSN: 9.5±1.4% vs 3.9±1.7%, P<0.05) was attenuated in A5-lesioned rats compared to controls. Our data demonstrated that A5 noradrenergic neurons are part of the circuitry recruited for the processing of sympathetic response to hypoxia and hypercapnia in unanesthetized conditions., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

The pontine nuclei (PN) are the largest of the precerebellar nuclei, neuronal assemblies in the hindbrain providing principal input to the cerebellum. The PN are predominantly innervated by the cerebral cortex and project as mossy fibers to the cerebellar hemispheres. Here, we comprehensively review the development of the PN from specification to migration, nucleogenesis and circuit formation. PN neurons originate at the posterior rhombic lip and migrate tangentially crossing several rhombomere derived territories to reach their final position in ventral part of the pons. The developing PN provide a classical example of tangential neuronal migration and a study system for understanding its molecular underpinnings. We anticipate that understanding the mechanisms of PN migration and assembly will also permit a deeper understanding of the molecular and cellular basis of cortico-cerebellar circuit formation and function.

Urine release (micturition) serves an essential physiological function as well as a critical role in social communication in many animals. Here, we show a combined effect of olfaction and social hierarchy on micturition patterns in adult male mice, confirming the existence of a micturition control center that integrates pro- and anti-micturition cues. Furthermore, we demonstrate that a cluster of neurons expressing corticotropin-releasing hormone (Crh) in the pontine micturition center (PMC) is electrophysiologically distinct from their Crh-negative neighbors and sends glutamatergic projections to the spinal cord. The activity of PMC Crh-expressing neurons correlates with and is sufficient to drive bladder contraction, and when silenced impairs micturition behavior. These neurons receive convergent input from widespread higher brain areas that are capable of carrying diverse pro- and anti-micturition signals, and whose activity modulates hierarchy-dependent micturition. Taken together, our results indicate that PMC Crh-expressing neurons are likely the integration center for context-dependent micturition behavior., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Kölliker-Fuse (KF) region, located in the dorsolateral pons, projects to several brainstem areas involved in respiratory regulation, including the chemoreceptor neurons within the retrotrapezoid nucleus (RTN). Several lines of evidence indicate that the pontine KF region plays an important role in the control of the upper airways for the maintenance of appropriate airflow to and from the lungs. Specifically, we hypothesized that the KF region is involved in mediating the response of the hypoglossal motor activity to central respiratory chemoreflex activation and to stimulation of the chemoreceptor neurons within the RTN region. To test this hypothesis, we combined immunohistochemistry and physiological experiments. We found that in the KF, the majority of biotinylated dextran amine (BDA)-labeled axonal varicosities contained detectable levels of vesicular glutamate transporter-2 (VGLUT2), but few contained glutamic acid decarboxylase-67 (GAD67). The majority of the RTN neurons that were FluorGold (FG)-immunoreactive (i.e., projected to the KF) contained hypercapnia-induced Fos, but did not express tyrosine hydroxylase. In urethane-anesthetized sino-aortic denervated and vagotomized male Wistar rats, hypercapnia (10% CO2) or N-methyl-d-aspartate (NMDA) injection (0.1mM) in the RTN increased diaphragm (DiaEMG) and genioglossus muscle (GGEMG) activities and elicited abdominal (AbdEMG) activity. Bilateral injection of muscimol (GABA-A agonist; 2mM) into the KF region reduced the increase in DiaEMG and GGEMG produced by hypercapnia or NMDA into the RTN. Our data suggest that activation of chemoreceptor neurons in the RTN produces a significant increase in the genioglossus muscle activity and the excitatory pathway is dependent on the neurons located in the dorsolateral pontine KF region., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

The pons controls crucial sensorimotor and autonomic functions. In humans, it grows sixfold postnatally and is a site of paediatric gliomas; however, the mechanisms of pontine growth remain poorly understood. We show that the murine pons quadruples in volume postnatally; growth is fastest during postnatal days 0-4 (P0-P4), preceding most myelination. We identify three postnatal proliferative compartments: ventricular, midline and parenchymal. We find no evidence of postnatal neurogenesis in the pons, but each progenitor compartment produces new astroglia and oligodendroglia; the latter expand 10- to 18-fold postnatally, and are derived mostly from the parenchyma. Nearly all parenchymal progenitors at P4 are Sox2(+)Olig2(+), but by P8 a Sox2(-) subpopulation emerges, suggesting a lineage progression from Sox2(+) 'early' to Sox2(-) 'late' oligodendrocyte progenitor. Fate mapping reveals that >90% of adult oligodendrocytes derive from P2-P3 Sox2(+) progenitors. These results demonstrate the importance of postnatal Sox2(+)Olig2(+) progenitors in pontine growth and oligodendrogenesis.

The cerebellum receives extensive disynaptic input from the neocortex via the basal pontine nuclei, the neurons of which send mossy fiber (MF) axons to the granule cell layer of the contralateral cerebellar hemisphere. Although this cortico-cerebellar circuit has been implicated in tasks such as sensory discrimination and motor learning, little is known about the potential role of MF morphological plasticity in the function of the cerebellar granule cell layer. To address this issue, we labeled MFs with EGFP via viral infection of the basal pons in adult rats and performed in vivo two-photon imaging of MFs in Crus I/II of the cerebellar hemisphere over a period of several weeks. Following the acquisition of baseline images, animals were housed in control, enriched, or deprived sensory environments. Morphological dynamics were assessed by tracing MF axons and their terminals, and by tracking the stability of filopodia arising from MF terminal rosettes. MF axons and terminals were found to be remarkably stable. Parameters derived neither from measurements of axonal arbor geometry nor from the morphology of individual rosettes and their filopodial extensions significantly changed under control conditions over 4 weeks of imaging. Increasing whisker stimulation by manipulating the sensory environment or decreasing such stimulation by whisker trimming also failed to alter MF structure. Our studies indicate that pontine MF axons projecting to Crus I/II in adult rats do not undergo significant structural rearrangements over the course of weeks, and that this stability is not altered by the sustained manipulation of whisker sensorimotor experience.

Although it has been proposed that neurons that contain both acetylcholine (ACh) and γ-aminobutyric acid (GABA) are present in the prepositus hypoglossi nucleus (PHN), these neurons have not been characterized because of the difficulty in identifying them. In the present study, PHN neurons that express both choline acetyltransferase and the vesicular GABA transporter (VGAT) were identified using double-transgenic rats, in which the cholinergic and inhibitory neurons express the fluorescent proteins tdTomato and Venus, respectively. To characterize the neurons that express both tdTomato and Venus (D+ neurons), the afterhyperpolarization (AHP) profiles and firing patterns of these neurons were investigated via whole-cell recordings of brainstem slice preparations. Regarding the three AHP profiles and four firing patterns that the D+ neurons exhibited, an AHP with an afterdepolarization and a firing pattern that exhibited a delay in the generation of the first spike were the preferential properties of these neurons. In the three morphological types classified, the multipolar type that exhibited radiating dendrites was predominant among the D+ neurons. Immunocytochemical analysis revealed that the VGAT-immunopositive axonal boutons that expressed tdTomato were primarily located in the dorsal cap of inferior olive (IO) and the PHN. Although the PHN receives cholinergic inputs from the pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus, D+ neurons were absent from these brain areas. Together, these results suggest that PHN neurons that co-express ACh and GABA exhibit specific electrophysiological and morphological properties, and innervate the dorsal cap of the IO and the PHN., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Layer 5 pyramidal neurons (L5PNs) in the mouse prefrontal cortex respond to serotonin (5-HT) according to their long-distance axonal projections; 5-HT1A (1A) receptors mediate inhibitory responses in corticopontine (CPn) L5PNs, while 5-HT2A (2A) receptors can enhance action potential (AP) output in callosal/commissural (COM) L5PNs, either directly (in "COM-excited" neurons), or following brief 1A-mediated inhibition (in "COM-biphasic" neurons). Here we compare the impact of 5-HT on the excitability of CPn and COM L5PNs experiencing variable excitatory drive produced by current injection (DC current or simulated synaptic current) or with exogenous glutamate. 5-HT delivered at resting membrane potentials, or paired with subthreshold depolarizing input, hyperpolarized CPn and COM-biphasic L5PNs and failed to promote AP generation in COM-excited L5PNs. Conversely, when paired with suprathreshold excitatory drive generating multiple APs, 5-HT suppressed AP output in CPn L5PNs, enhanced AP generation in COM-excited L5PNs, and generated variable responses in COM-biphasic L5PNs. While COM-excited neurons failed to respond to 5-HT in the presence of a 2A receptor antagonist, 32% of CPn neurons exhibited 2A-dependent excitation following blockade of 1A receptors. The presence of pharmacologically revealed 2A receptors in CPn L5PNs was correlated with the duration of 1A-mediated inhibition, yet biphasic excitatory responses to 5-HT were never observed, even when 5-HT was paired with strong excitatory drive. Our results suggest that 2A receptors selectively amplify the output of COM L5PNs experiencing suprathreshold excitatory drive, while shaping the duration of 1A-mediated inhibition in a subset of CPn L5PNs. Activity-dependent serotonergic excitation of COM L5PNs, combined with 1A-mediated inhibition of CPn and COM-biphasic L5PNs, may facilitate executive function by focusing network activity within cortical circuits subserving the most appropriate behavioral output.

The ventral nucleus of the lateral lemniscus (VNLL) is an important nucleus in the central auditory pathway which connects the lower brainstem and the midbrain inferior colliculus (IC). Previous studies have demonstrated that neurons in the VNLL could respond to sound signal parameters. Frequency tuning curves (FTCs) of VNLL neurons are generally wider than FTCs of IC neurons, suggesting that the VNLL does not enhance abilities of frequency discrimination and coding. Two types of rate-intensity functions (RIFs) are found in the VNLL: monotonic and non-monotonic RIFs. Intensity-tuning of VNLL neurons are affected by the temporal firing patterns during processing and encoding intensity. There are multiple temporal firing patterns in VNLL neurons. Onset pattern has a precise timing characteristic which is well suited to encode temporal features of stimuli, and also very important to animal behavior including bat's echolocation. The VNLL accepts inputs from lower nuclei, uploads glycine inhibitory outputs to IC, and modulates response characteristics generating and acoustic signal processing of IC neurons. Recent research suggests that fast inhibitory projection from the VNLL may delay the first spike latency of IC neurons, and the delayed inhibitory projection from the VNLL may mediate the temporal firing patterns of IC neurons. But how inhibitory inputs from the VNLL integrate in IC, and how inhibitory inputs from the VNLL enhance the ability of detecting sound signal of IC neurons are not very clear and need more direct evidence at the level of neurons. These questions will help further understand the role of upload during IC processes acoustic signal, which are our research target in the future. This article reviews the current literature regarding the roles of the VNLL in sound signal processing and the auditory ascending transmission, including advances in the relevant research in our laboratory.

Posttetanic potentiation (PTP) is a widely observed form of short-term plasticity lasting for tens of seconds after high-frequency stimulation. Here we show that although protein kinase C (PKC) mediates PTP at the calyx of Held synapse in the auditory brainstem before and after hearing onset, PTP is produced primarily by an increased probability of release (p) before hearing onset, and by an increased readily releasable pool of vesicles (RRP) thereafter. We find that these mechanistic differences, which have distinct functional consequences, reflect unexpected differential actions of closely related calcium-dependent PKC isoforms. Prior to hearing onset, when PKCγ and PKCβ are both present, PKCγ mediates PTP by increasing p and partially suppressing PKCβ actions. After hearing onset, PKCγ is absent and PKCβ produces PTP by increasing RRP. In hearing animals, virally expressed PKCγ overrides PKCβ to produce PTP by increasing p. Thus, two similar PKC isoforms mediate PTP in distinctly different ways., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Primates explore a visual scene through a succession of saccades. Much of what is known about the neural circuitry that generates these movements has come from neurophysiological studies using subjects with their heads restrained. Horizontal saccades and the horizontal components of oblique saccades are associated with high-frequency bursts of spikes in medium-lead burst neurons (MLBs) and long-lead burst neurons (LLBNs) in the paramedian pontine reticular formation. For LLBNs, the high-frequency burst is preceded by a low-frequency prelude that begins 12-150 ms before saccade onset. In terms of the lead time between the onset of prelude activity and saccade onset, the anatomical projections, and the movement field characteristics, LLBNs are a heterogeneous group of neurons. Whether this heterogeneity is endemic of multiple functional subclasses is an open question. One possibility is that some may carry signals related to head movement. We recorded from LLBNs while monkeys performed head-unrestrained gaze shifts, during which the kinematics of the eye and head components were dissociable. Many cells had peak firing rates that never exceeded 200 spikes/s for gaze shifts of any vector. The activity of these low-frequency cells often persisted beyond the end of the gaze shift and was usually related to head-movement kinematics. A subset was tested during head-unrestrained pursuit and showed clear modulation in the absence of saccades. These "low-frequency" cells were intermingled with MLBs and traditional LLBNs and may represent a separate functional class carrying signals related to head movement.

Nuclei in the central nervous system are 3D aggregates of neurons that have common physiological properties, functionalities, and connectivities. To form specific nuclei, neurons migrate from their birthplace towards the presumptive nuclear region where they change their dynamics to aggregate and rearrange into a distinct 3D structure, a process that we term nucleogenesis. Nuclei, together with the laminar structure, form the basic cytoarchitectonic unit for information processing. However, in contrast to much-studied laminar structures, the neuronal dynamics that contribute to the aggregation process to form nuclei are poorly understood. Here, we analyze nucleogenesis by observing the mouse precerebellar pontine nucleus (PN), and provide the first 4D view of nucleogenesis by tracking neuronal behaviors along the three spatial axes over time. Early- and late-born PN neurons were labeled by in utero electroporation and their behaviors on cultured brain slices were recorded by time-lapse imaging. We find that when PN neurons migrate medially into the nuclear region, many of them switch to migrate radially and laterally, to populate the dorsal and lateral PN regions, respectively. The tendency to switch to radial migration is much less in later-born neurons, whereas that to switch to lateral migration is comparable between the two groups. In contrast to the radial and mediolateral axes, very few PN neurons switch to migrate rostrocaudally. These results could thus provide a framework for understanding the mechanisms that regulate this complex yet important process., (© 2013 Wiley Periodicals, Inc.)

Diphtheria toxin-mediated, acute ablation of hypothalamic neurons expressing agouti-related protein (AgRP) in adult mice leads to anorexia and starvation within 7 d that is caused by hyperactivity of neurons within the parabrachial nucleus (PBN). Because NMDA glutamate receptors are involved in various synaptic plasticity-based behavioral modifications, we hypothesized that modulation of the NR2A and NR2B subunits of the NMDA receptor in PBN neurons could contribute to the anorexia phenotype. We observed by Western blot analyses that ablation of AgRP neurons results in enhanced expression of NR2B along with a modest suppression of NR2A. Interestingly, systemic administration of LiCl in a critical time window before AgRP neuron ablation abolished the anorectic response. LiCl treatment suppressed NR2B levels in the PBN and ameliorated the local Fos induction that is associated with anorexia. This protective role of LiCl on feeding was blunted in vagotomized mice. Chronic infusion of RO25-6981, a selective NR2B inhibitor, into the PBN recapitulated the role of LiCl in maintaining feeding after AgRP neuron ablation. We suggest that the accumulation of NR2B subunits in the PBN contributes to aphagia in response to AgRP neuron ablation and may be involved in other forms of anorexia.

The present study investigated the projection from the paralemniscal nucleus (PL) to the spinal cord in the mouse by injecting the retrograde tracer fluoro-gold to different levels of the spinal cord and injecting the anterograde tracer biotinylated dextran amine into PL. We found that PL projects to the entire spinal cord with obvious contralateral predominance--420 neurons projected to the contralateral cervical cord and 270 to the contralateral lumbar cord. Fibers from PL descended in the dorsolateral funiculus on the contralateral side and terminated in laminae 5, 6, 7, and to a lesser extent in the dorsal and ventral horns. A smaller number of fibers also descended in the ventral funiculus on the ipsilateral side and terminated in laminae 7, 8 and, to a lesser extent in lamina 9. The present study is the first demonstration of the PL fiber termination in the spinal cord in mammals. The PL projection to the spinal cord may be involved in vocalization and locomotion.

Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells. The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved. Using cell-type-specific projection mapping with synaptic resolution, we observed the convergence of separate sensory (upper body proprioceptive) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex. These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function. We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas. Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control. DOI:http://dx.doi.org/10.7554/eLife.00400.001.

We have found a previously unreported precerebellar nucleus located among the emerging fibers of the motor root of the trigeminal nerve in the mouse, which we have called the interfascicular trigeminal nucleus (IF5). This nucleus had previously been named the tensor tympani part of the motor trigeminal nucleus (5TT) in rodent brain atlases, because it was thought to be a subset of small motor neurons of the motor trigeminal nucleus innervating the tensor tympani muscle. However, following injection of retrograde tracer in the cerebellum, the labeled neurons in IF5 were found to be choline acetyltransferase (ChAT) negative, indicating that they are not motor neurons. The cells of IF5 are strongly labeled in mice from Wnt1Cre and Atoh1 CreER lineage fate mapping, in common with the major precerebellar nuclei that arise from the rhombic lip and that issue mossy fibers. Analysis of sections from mouse Hoxa3, Hoxb1, and Egr2 Cre labeled lineages shows that the neurons of IF5 arise from rhombomeres caudal to rhombomere 4, most likely from rhombomeres 6-8. We conclude that IF5 is a significant precerebellar nucleus in the mouse that shares developmental gene expression characteristics with mossy fiber precerebellar nuclei that arise from the caudal rhombic lip., (Copyright © 2012 Wiley Periodicals, Inc.)

Pontine A5, A6 (locus coeruleus) and A7 cell groups provide noradrenergic innervation of the spinal cord. Here, we assessed whether activation of α(2) -adrenoceptors in A7 influences peripheral nerve injury-induced hypersensitivity in the rat, and whether spinal α(2) -adrenoceptors mediate the descending effect. Fadolmidine, an α(2) -adrenoceptor agonist that because of its pharmacokinetic properties has a limited spread from the injection site, was microinjected through a chronic guide cannula into A7 or for comparison into A6 ipsilateral to the nerve injury. Moreover, atipamezole, an α(2) -adrenoceptor antagonist, was injected intrathecally in an attempt to reverse the possible antihypersensitivity effect. Tactile hypersensitivity was assessed in the injured limb by determining limb withdrawal evoked by calibrated monofilaments, mechanical hyperalgesia by determining withdrawal evoked by noxious mechanical stimulation of the paw, and thermal nociception by assessing heat-induced withdrawal of the intact tail. Fadolmidine (1.0 or 3.0 μg) in A7, but not in A6, produced a tactile antihypersensitivity effect. Intrathecal atipamezole (5 μg) reversed the tactile antihypersensitivity effect by fadolmidine in A7. Atipamezole alone (5.0 μg) intrathecally or in A7 failed to influence tactile hypersensitivity. Fadolmidine in A7 failed to influence mechanical hyperalgesia or heat nociception at doses that produced a tactile antihypersensitivity effect. We propose that tonic noradrenergic drive of A6 by A7 promotes neuropathic hypersensitivity by suppressing descending noradrenergic inhibition originating in A6. Consequently, the activation of inhibitory α(2) -adrenoceptors within the pontine A7 cell group suppresses neuropathic hypersensitivity by disinhibiting A6 and its descending noradrenergic pathways acting on spinal α(2) -adrenoceptors., (© 2012 The Authors Basic & Clinical Pharmacology & Toxicology © 2012 Nordic Pharmacological Society.)

We investigated the role of histone methyltransferase Ezh2 in tangential migration of mouse precerebellar pontine nuclei, the main relay between neocortex and cerebellum. By counteracting the sonic hedgehog pathway, Ezh2 represses Netrin1 in dorsal hindbrain, which allows normal pontine neuron migration. In Ezh2 mutants, ectopic Netrin1 derepression results in abnormal migration and supernumerary nuclei integrating in brain circuitry. Moreover, intrinsic topographic organization of pontine nuclei according to rostrocaudal progenitor origin is maintained throughout migration and correlates with patterned cortical input. Ezh2 maintains spatially restricted Hox expression, which, in turn, regulates differential expression of the repulsive receptor Unc5b in migrating neurons; together, they generate subsets with distinct responsiveness to environmental Netrin1. Thus, Ezh2-dependent epigenetic regulation of intrinsic and extrinsic transcriptional programs controls topographic neuronal guidance and connectivity in the cortico-ponto-cerebellar pathway.

Precise temporal and spatial sequences of synaptogenesis were demonstrated in 172 human foetuses and neonates post-mortem in transverse paraffin sections of pons and cerebellar vermis and hemispheres, using synaptophysin immunoreactivity of this protein of synaptic vesicular walls. The pontine nuclei exhibit a transitory patchy pattern not predicted from the uniform histology and reminiscent of the corpus striatum; synaptic vesicle reactivity appears at 20 weeks and is uniform by 34 weeks. In the cerebellar cortex, the vermis matures sooner than the cerebellar hemispheres and the paravermal portions earlier than the lateral folia. The earliest synapses occur around the somata of Purkinje neurons and later in the internal granular layer, but synaptic glomeruli are not well formed until after 26 weeks. The normal patterns here shown, together with earlier data of the Guillain-Mollaret triangle, provide controls for the interpretation of synaptic delay or precociousness and other pathological patterns in malformations, genetic/metabolic conditions and prenatal acquired insults affecting the human foetus., (Copyright © 2013 S. Karger AG, Basel.)

Dr DeGroat and Wickens has reviewed the central neural mechanisms controlling the lower urinary tract with a major focus on the brain stem circuitry that mediates the switch-like characteristics of micturition, in particular the periaqueductal grey and the pontine micturition centre (de 2012). The review culminates in a computer model of how the brainstem switch operates in animals in which forebrain influences on micturition have been removed by decerebration. In this complementary paper, we review the mechanisms of forebrain involvement in the voluntary control of human micturition and the maintenance of continence with evidence based heavily on the results of functional brain imaging experiments., (© 2012 The Authors Acta Physiologica © 2012 Scandinavian Physiological Society.)

Stimulation of the posterior hypothalamic area (PH) produces antinociception in rats and humans, but the precise mechanisms are unknown. The PH forms anatomical connections with the parabrachial area, which contains the pontine A7 catecholamine cell group, a group of spinally projecting noradrenergic neurons known to produce antinociception in the dorsal horn. The aim of the present study was to determine whether PH-induced antinociception is mediated in part through connections with the A7 cell group in female Sprague-Dawley rats, as measured by the tail flick and foot withdrawal latency. Stimulation of the PH with the cholinergic agonist carbachol (125 nmol) produced antinociception that was blocked by pretreatment with atropine sulfate. Intrathecal injection of the α(2)-adrenoceptor antagonist yohimbine reversed PH-induced antinociception, but the α(1)-adrenoceptor antagonist WB4101 facilitated antinociception. Intrathecal injection of normal saline had no effect. In a separate experiment, cobalt chloride, which reversibly arrests synaptic activity, was microinjected into the A7 cell group and blocked PH-induced antinociception. These findings provide evidence that the PH modulates nociception in part through connections with the A7 catecholamine cell group through opposing effects. Antinociception occurs from actions at α(2)-adrenoceptors in the dorsal horn, while concurrent hyperalgesia occurs from actions of norepinephrine at α(1)-adrenoceptors. This hyperalgesic response likely attenuates antinociception from PH stimulation., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neural activity in several limbic areas varies as a function of the animal's head direction (HD) in the horizontal plane. Lesions of the vestibular periphery abolish this HD cell signal, suggesting an essential role for vestibular afference in HD signal generation. The organization of brain stem pathways conveying vestibular information to the HD circuit is poorly understood; however, recent anatomical work has identified the supragenual nucleus (SGN) as a putative relay. To test this hypothesis, we made lesions of the SGN in rats and screened for HD cells in the anterodorsal thalamus. In animals with complete bilateral lesions, the overall number of HD cells was significantly reduced relative to control animals. In animals with unilateral lesions of the SGN, directional activity was present, but the preferred firing directions of these cells were unstable and less influenced by the rotation of an environmental landmark. In addition, we found that preferred directions displayed large directional shifts when animals foraged for food in a darkened environment and when they were navigating from a familiar environment to a novel one, suggesting that the SGN plays a critical role in projecting essential self-motion (idiothetic) information to the HD cell circuit.

The stress-related neuropeptide, corticotropin-releasing factor (CRF), is prominent in neurons of the pontine micturition center, Barrington's nucleus. These neurons co-innervate spinal preganglionic neurons that control the bladder, and locus coeruleus (LC) neurons that provide norepinephrine innervation throughout the brain. Adeno-associated viral (AAV) vector-mediated transfer of CRF cDNA was used to increase CRF expression in Barrington's nucleus neurons and investigate the impact of a gain of function in Barrington's nucleus spinal and LC projections. AAV transfer of the reverse CRF cDNA sequence served as the control. Bladder urodynamics and behavior were assessed 4 weeks after vector injection into Barrington's nucleus. Rats with bilateral injections of AAV-CRF cDNA into Barrington's nucleus had immunohistochemical evidence of CRF overexpression in neurons and transport to the spinal cord and LC. The bladder : body weight ratio was greater and micturition pressure was less in these rats compared with controls, consistent with an inhibitory influence on bladder function. Other indices of urodynamic function were not altered. CRF innervation of the LC was increased in rats with bilateral Barrington's nucleus injections of AAV-CRF cDNA, and this was associated with increased burying behavior, an endpoint of LC activation by CRF. The results provide immunohistochemical evidence for viral vector-induced CRF overexpression in Barrington's nucleus neurons and underscore the ability of AAV vector-mediated transfer to increase CRF function in selective circuits. The findings support an inhibitory influence of CRF in Barrington's nucleus regulation of the bladder and an excitatory influence on the brain norepinephrine system that translates to behavioral activation., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The cuneiform (CnF) and Kolliker-Fuse (KF) nuclei are implicated in several functions including regulation of cardiovascular system and pain modulation. The KF also is a potential candidate for relaying the CnF cardiovascular responses to the rostral ventrolateral medulla (RVLM). In a previous study we showed that blockade of the KF strongly attenuated the short responses and moderately attenuated the long responses to glutamate microinjection into the CnF, suggesting that the cardiovascular effects of the CnF, especially the short responses, were mediated by the KF. In the present study the cellular basis of the cardiovascular responses of the CnF and possible role of the KF in relaying them to the RVLM were explored. In one group, l-glutamate was microinjected in the CnF and the cardiovascular responses were recorded. In another group the single unit responses of the KF to l-glutamate injection into the CnF were recorded. Our results showed that chemical stimulation of the CnF with glutamate produced mainly excitatory cardiovascular and single unit responses and a minority of mixed (excitatory and inhibitory) responses. In about one fourth of the cases there were no responses to stimulation. Various patterns of each group were presented and compared between cardiovascular and single unit responses. Similarities were found between cardiovascular and single unit response patterns, suggesting a significant role of KF neurons in mediating the CnF cardiovascular responses to the RVLM., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: The abducens nucleus directly innervates the lateral rectus muscle and plays a role in controlling conjugate horizontal eye movements. Although the neuronal cytoarchitecture of the abducens nucleus has been extensively investigated in various species of vertebrates, few studies have been undertaken in humans, especially in fetuses or neonates., Design/subjects: We examined 12 human brains from preterm infants aged 20-43 postmenstrual weeks to document the histology and morphometry of the abducens nucleus. The brain was processed into celloidin-embedded serial sections stained with the Klüver-Barrera and other conventional methods., Results: The nucleus was identified as a mass of cells as early as 20 weeks. Its neurons were clearly distinguished from glial cells due to droplet-like, clear nuclei containing prominent nucleoli and surrounded by a basophilic perikaryon. Neurons of various sizes and shapes were intermingled within the nucleus, although larger neurons were located towards the center of the nucleus. Immature granular or reticular Nissl bodies were seen at 20-21 weeks. Tigroid, coarse Nissl bodies appeared around 28-29 weeks in larger neurons, although in smaller neurons Nissl bodies were dispersed or concentrated peripherally. Morphometric results were: (1) the nuclear volume exponentially increased with age between 20 and 43 weeks; (2) the histograms of neuronal profile areas showed a non-normal distribution trailing toward the right and widening with age; (3) the geometric average of neuronal profile areas increased linearly with age., Conclusion: Our study suggests that the human abducens nucleus enlarges more quickly toward the end of gestation, and comprises heterogeneous groups of neurons., (Copyright © 2012 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

The parabrachial nuclei (PbN), the second central relay for the gustatory pathway, transfers taste information to various forebrain gustatory nuclei and to the gustatory cortex. The nucleus accumbens is one of the critical neural substrates of the reward system, and the nucleus accumbens shell region (NAcSh) is associated with feeding behavior. Taste-evoked neuronal responses of PbN neurons are modulated by descending projections from the gustatory nuclei in the forebrain. In the present study, we investigated whether taste-responsive neurons in the PbN project to the NAcSh and whether pontine gustatory neurons are subject to modulatory influence from the NAcSh in urethane-anesthetized hamsters. Extracellular single-unit activity was recorded in the PbN, and taste responses were confirmed by the delivery of 32 mM sucrose, NaCl, quinine hydrochloride, and 3.2 mM citric acid to the anterior tongue. The NAcSh was then stimulated (0.5 ms, ≤100 μA) bilaterally using concentric bipolar stimulating electrodes. A total of 98 taste neurons were recorded from the PbN. Eighteen neurons were antidromically invaded from the NAcSh, mostly the ipsilateral NAcSh (n = 16). Stimulation of the ipsilateral and contralateral NAcSh suppressed the neuronal activity of 88 and 55 neurons, respectively; 52 cells were affected bilaterally. In a subset of pontine neurons tested, electrical stimulation of the NAcSh during taste stimulation also suppressed taste-evoked neuronal firing. These results demonstrated that taste-responsive neurons in the PbN not only project to the NAcSh but also are under substantial descending inhibitory influence from the bilateral NAcSh.

The parabrachial nucleus (PB) is a brainstem cell group that receives a strong input from the nucleus tractus solitarius regarding the physiological status of the internal organs and sends efferent projections throughout the forebrain. Since the neuroanatomical organization of the PB remains unclear, our first step was to use specific antibodies against two neural lineage transcription factors: Forkhead box protein2 (FoxP2) and LIM homeodomain transcription factor 1 beta (Lmx1b) to define the PB in adult rats. This allowed us to construct a cytoarchitectonic PB map based on the distribution of neurons that constitutively express these two transcription factors. Second, the in situ hybridization method combined with immunohistochemistry demonstrated that mRNA for glutamate vesicular transporter Vglut2 (Slc17a6) was present in most of the Lmx1b+ and FoxP2+ parabrachial neurons, indicating these neurons use glutamate as a transmitter. Third, conscious rats were maintained in a hypotensive or hypertensive state for 2h, and then, their brainstems were prepared by the standard c-Fos method which is a measure of neuronal activity. Both hypotension and hypertension resulted in c-Fos activation of Lmx1b+ neurons in the external lateral-outer subdivision of the PB (PBel-outer). Hypotension, but not hypertension, caused c-Fos activity in the FoxP2+ neurons of the central lateral PB (PBcl) subnucleus. The Kölliker-Fuse nucleus as well as the lateral crescent PB and rostral-most part of the PBcl contain neurons that co-express FoxP2+ and Lmx1b+, but none of these were activated after blood pressure changes. Salt-sensitive FoxP2 neurons in the pre-locus coeruleus and PBel-inner were not c-Fos activated following blood pressure changes. In summary, the present study shows that the PBel-outer and PBcl subnuclei originate from two different neural progenitors, contain glutamatergic neurons, and are affected by blood pressure changes, with the PBel-outer reacting to both hypo- and hypertension, and the PBcl signaling only hypotensive changes., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

During exercise, intense brain activity orchestrates an increase in muscle tension. Additionally, there is an increase in cardiac output and ventilation to compensate the increased metabolic demand of muscle activity and to facilitate the removal of CO(2) from and the delivery of O(2) to tissues. Here we tested the hypothesis that a subset of pontomedullary and hypothalamic neurons could be activated during dynamic acute exercise. Male Wistar rats (250-350 g) were divided into an exercise group (n=12) that ran on a treadmill and a no-exercise group (n=7). Immunohistochemistry of pontomedullary and hypothalamic sections to identify activation (c-Fos expression) of cardiorespiratory areas showed that the no-exercise rats exhibited minimal Fos expression. In contrast, there was intense activation of the nucleus of the solitary tract, the ventrolateral medulla (including the presumed central chemoreceptor neurons in the retrotrapezoid/parafacial region), the lateral parabrachial nucleus, the Kölliker-Fuse region, the perifornical region, which includes the perifornical area and the lateral hypothalamus, the dorsal medial hypothalamus, and the paraventricular nucleus of the hypothalamus after running exercise. Additionally, we observed Fos immunoreactivity in catecholaminergic neurons within the ventrolateral medulla (C1 region) without Fos expression in the A2, A5 and A7 neurons. In summary, we show for the first time that after acute exercise there is an intense activation of brain areas crucial for cardiorespiratory control. Possible involvement of the central command mechanism should be considered. Our results suggest whole brain-specific mobilization to correct and compensate the homeostatic changes produced by acute exercise., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Gudden's tegmental nuclei provide major inputs to the rodent mammillary bodies, where they are thought to be important for learning and navigation. Comparable projections have yet to be described in the primate brain, where part of the problem has been in effectively delineating these nuclei. Immunohistochemical staining of tissue from a series of macaque monkeys (Macaca mulatta) showed that cells in the region of both the ventral and dorsal tegmental nuclei selectively stain for parvalbumin, thus helping to reveal these nuclei. These same tegmental nuclei were not selectively revealed when tissue was stained for SMI32, acetylcholinesterase, calbindin, or calretinin. In a parallel study, horseradish peroxidase was injected into the mammillary bodies of five cynomolgus monkeys (Macaca fascicularis). Retrogradely labeled neurons were consistently found in the three subdivisions of the ventral tegmental nucleus of Gudden, which are located immediately below, within, and above the medial longitudinal fasciculus. Further projections to the mammillary body region arose from cells in the anterior tegmental nucleus, which appears to be a rostral continuation of the infrafascicular part of the ventral tegmental nucleus. In the dorsal tegmental nucleus of Gudden, labeled cells were most evident when the tracer injection was more laterally placed in the mammillary bodies, consistent with a projection to the lateral mammillary nucleus. The present study not only demonstrates that the primate mammillary bodies receive parallel inputs from the dorsal and ventral tegmental nuclei of Gudden, but also helps to confirm the extent of these poorly distinguished nuclei in the monkey brain.

The pontine nuclei (PN) are the major intermediary elements in the corticopontocerebellar pathway. Here we asked if the PN may help to adapt the spatial reference frames used by cerebrocortical neurons involved in the sensory guidance of movement to a format potentially more appropriate for the cerebellum. To this end, we studied movement-related neurons in the dorsal PN (DPN) of monkeys, most probably projecting to the cerebellum, executing fixed vector saccades or, alternatively, fixed vector hand reaches from different starting positions. The 83 task-related neurons considered fired movement-related bursts before saccades (saccade-related) or before hand movements (hand movement-related). About 40% of the SR neurons were "oculocentric," whereas the others were modulated by eye starting position. A third of the HMR neurons encoded hand reaches in hand-centered coordinates, whereas the remainder exhibited different types of dependencies on starting positions, reminiscent in general of cortical responses. All in all, pontine reference frames for the sensory guidance of movement seem to be very similar to those in cortex. Specifically, the frequency of orbital position gain fields of SR neurons is identical in the DPN and in one of their major cortical inputs, lateral intraparietal area (LIP).

Changes in the direction of the line of sight (gaze) allow successive sampling of the visual environment. Saccadic eye movements accomplish this goal when the head does not move. Medium-lead burst neurons (MLBs) in the paramedian pontine reticular formation (PPRF) discharge a high frequency burst of action potentials starting ~12 ms before the saccade begins. A subgroup of MLBs rostral of abducens nucleus monosynaptically excites oculomotor neurons. The number of spikes in the presaccadic burst is correlated with the amplitude of the horizontal component of the saccade, and the peak discharge rate is correlated with peak eye velocity. During head-unrestrained gaze shifts, a linear relationship between the number of action potentials in MLB bursts and gaze (but not eye) amplitude has been reported. The anatomical connection of MLBs to motor neurons and the similarity between the phasic motor neuron burst and MLB discharge have raised questions about the usefulness of counting spikes in MLBs to determine their role in eye-head coordination. We investigated this issue using a behavioral technique that permits a dissociation of eye movement amplitude and duration during constant vector gaze shifts. Surprisingly, during gaze shifts of constant amplitude and direction, we observe a nearly linear, positive correlation between saccade duration and spike number associated with a negative correlation between spike number and saccade amplitude. These data constrain models of the oculomotor controller and may further define the time-dependence of hypothesized neural integration in this system.

This study was performed to understand the anatomical substrates for Kölliker-Fuse nucleus (KFN) modulation of respiratory-related tongue movement. After application of cholera toxin B subunit (CTb) to the medial branch of the hypoglossal nerve (HGn) and injection of biotinylated dextran amine (BDA) into the KFN ipsilaterally, an overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found in the ventral compartment of the hypoglossal nucleus (HGN) ipsilateral to the application and injection sites. At the electron microscopic level, the BDA-labeled terminals made asymmetrical synaptic contacts predominantly with dendrites of the HGN neurons, some of which were labeled with CTb. Using retrograde tracing combined with in situ hybridization, we demonstrated that almost all the KFN neurons sending their axons to the HGN were positive for vesicular glutamate transporter (VGLUT) 2 mRNA but not glutamic acid decarboxylase 67 mRNA. Using a combination of anterograde and retrograde tracing techniques and immunohistochemistry for VGLUT2, we further demonstrated that the KFN axon terminals with VGLUT2 immunoreactivity established close contact with the HGN motoneurons whose axons constitute the medial branch of the HGn. The present results suggest that glutamatergic KFN fibers may exert excitatory influence upon the HGN motoneurons sending their axons to the medial branch of the HGn for the control of protruder tongue muscles contraction to maintain airway patency during respiration., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The dorsal subcoeruleus nucleus (SubCD) is involved in generating two signs of rapid eye movement (REM) sleep: muscle atonia and ponto-geniculo-occipital (PGO) waves. We tested the hypothesis that single cell and/or population responses of SubCD neurons are capable of generating gamma frequency activity in response to intracellular stimulation or receptor agonist activation. Whole cell patch clamp recordings (immersion chamber) and population responses (interface chamber) were conducted on 9- to 20-day-old rat brain stem slices. All SubCD neurons (n = 103) fired at gamma frequency when subjected to depolarizing steps. Two statistically distinct populations of neurons were observed, which were distinguished by their high (>80 Hz, n = 24) versus low (35-80 Hz, n = 16) initial firing frequencies. Both cell types exhibited subthreshold oscillations in the gamma range (n = 43), which may underlie the gamma band firing properties of these neurons. The subthreshold oscillations were blocked by the sodium channel blockers tetrodotoxin (TTX, n = 21) extracellularly and N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314) intracellularly (n = 5), indicating they were sodium channel dependent. Gamma frequency subthreshold oscillations were observed in response to the nonspecific cholinergic receptor agonist carbachol (CAR, n = 11, d = 1.08) and the glutamate receptor agonists N-methyl-d-aspartic acid (NMDA, n = 12, d = 1.09) and kainic acid (KA, n = 13, d = 0.96), indicating that cholinergic and glutamatergic inputs may be involved in the activation of these subthreshold currents. Gamma band activity also was observed in population responses following application of CAR (n = 4, P < 0.05), NMDA (n = 4, P < 0.05) and KA (n = 4, P < 0.05). Voltage-sensitive, sodium channel-dependent gamma band activity appears to be a part of the intrinsic membrane properties of SubCD neurons.

Noradrenergic (NAergic) A7 neurons that project axonal terminals to the dorsal horn of the spinal cord to modulate nociceptive signaling are suggested to receive tonic inhibition from local GABAergic interneurons, which are under the regulation of descending analgesic pathways. In support of this argument, we presently report GABA(B) receptor (GABA(B)R)-mediated tonic inhibition of NAergic A7 neurons. Bath application of baclofen induced an outward current (I(Bac)) in NAergic A7 neurons that was blocked by CGP 54626, a GABA(B)R blocker. The I(Bac) was reversed at about -99 mV, displayed inward rectification, and was blocked by Ba(2+) or Tertipian-Q, showing it was mediated by G protein-activated inward-rectifying K(+) (GIRK) channels. Single-cell RT-PCR results suggested that GIRK1/3 heterotetramers might dominate functional GIRK channels in NAergic A7 neurons. Under conditions in which GABA(A) and glycine receptors were blocked, bath application of GABA inhibited the spontaneous firing of NAergic A7 neurons in a dose-dependent manner. Interestingly, CGP 54626 application not only blocked the effect of GABA but also increased the firing rate to 126.9% of the control level, showing that GABA(B)Rs were constitutively active at an ambient GABA concentration of 2.8 μM and inhibited NAergic A7 neurons. GABA(B)Rs were also found at presynaptic excitatory and inhibitory axonal terminals in the A7 area. Pharmacological activation of these GABA(B)Rs inhibited the release of neurotransmitters. No physiological role was found for GABA(B)Rs on excitatory terminals, whereas those on the inhibitory terminals were found to exert autoregulatory control of GABA release.

The calyx of Held synapse is a giant axosomatic synapse that has a fast relay function within the sound localization circuit of the brainstem. In the adult, each principal neuron of the medial nucleus of the trapezoid body (MNTB) is contacted by a single calyx terminal. In rodents, the calyx of Held synapse forms around the third postnatal day (P3). Here, we studied the developmental changes in the intrinsic excitability of the principal neurons during the first postnatal week by making whole-cell recordings from brainstem slices. In slices from P0-1 rats, about 20% of the principal neurons were spontaneously active, whereas after P3, no spontaneously active cells were observed. Already at P0, principal neurons received both glutamatergic and GABAergic/glycinergic inputs. The occurrence of spontaneous action potentials depended upon the presence of spontaneous glutamatergic inputs; summation of only a few quanta was enough to reach action potential threshold. The main cause for this high excitability was a high resting membrane resistance, which decreased at least four-fold during the first postnatal week. A relatively slow decay of synaptic currents and a relatively depolarized membrane potential may have contributed as well. We conclude that the decrease in the excitability of principal neurons in the MNTB matches the increase of the strength of the synaptic inputs resulting from the formation and maturation of the calyx of Held synapse during the first postnatal week. This decrease in excitability will make it progressively more difficult for non-calyceal inputs to trigger action potentials., (2010 Wiley Periodicals, Inc.)

In the present work we study the contribution of the chloride channel of the Cystic Fibrosis Transmembrane Regulator (CFTR) in the postsynaptic inhibition of somatic motoneurons during rapid-eye-movement (REM) sleep atonia. Postsynaptic inhibition of motoneurons is partially responsible for the atonia that occurs during REM sleep. Disfacilitation is an additional mechanism that lowers motoneuron excitability in this state. Postsynaptic inhibition is mediated by the release of glycine from synaptic terminals on motoneurons, and by GABA that plays a complementary role to that of glycine. In this work we look in brain stem motoneurons of neonatal rats at a mechanism unrelated to the actions of glycine, GABA or to disfacilitation which depends on the chloride channel of the CFTR. We studied the presence of CFTR by immunocytochemistry. In electrophysiological experiments utilizing whole cell recordings in in vitro slices we examined the consequences of blocking this chloride channel. The effects on motoneurons of the application of glycine, of the application of glibenclamide (a CFTR blocker) and again of glycine during the effects of glibenclamide were studied. Glycine produced an hyperpolarization, a decrease in motoneuron excitability and a decrease in input resistance, all characteristic changes of the postsynaptic inhibition produced by this neurotransmitter. Glibenclamide produced an increase in input resistance and in motoneurons' repetitive discharge as well as a shift in the equilibrium potential for chloride ions as indicated by the displacement of the reversal potential for glycinergic actions. In motoneurons treated with glibenclamide, glycine produced postsynaptic inhibition but this effect was smaller when compared to that elicited by glycine in control conditions. The fact that blocking of the CFTR-chloride channel in brain stem motoneurons influences glycinergic inhibition suggests that this channel may play a complementary role in the glycinergic inhibition that occurs during REM sleep., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Two specific groups of neurons in the dorsolateral pons are activated by dietary sodium deprivation. These two groups are the pre-locus coeruleus (pre-LC) and the inner subdivision of the external lateral parabrachial nucleus (PBel-inner). In each site, after rats are fed an extremely low-sodium diet for over a week, neurons increase their expression of an activity-induced transcription factor, c-Fos. Here, we confirm this observation and extend it by demonstrating that these two groups of neurons express a common marker gene, the constitutively-expressed transcription factor Forkhead box protein 2 (FoxP2). That is, virtually all of the c-Fos activated neurons in both regions also express FoxP2. The expression of FoxP2 by both these groups of neurons suggests that they are developmentally-related subsets derived from the same basic population. Given that FoxP2, unlike c-Fos, is expressed independent of sodium deprivation, this marker may be useful in future studies of the pre-LC and PBel-inner. The molecular definition of these neurons, which project to circuits in the forebrain that influence visceral, appetitive, and hedonic functions, may allow direct experimental exploration of the functional role of these circuits using genetic tools., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by polyglutamine-expanded ataxin-3. SCA3 neurodegeneration is found in the pontine nuclei and cerebellum. Polyglutamine-expanded ataxin-3-Q79 caused apoptotic death of cerebellar and pontine nuclei neurons by upregulating mRNA expression of pro-apoptotic Bax and activating mitochondria-mediated apoptotic cascade. Following various cellular stresses, transcription factor p53 promotes apoptotic neuronal death by enhancing the transcription of pro-apoptotic genes including Bax and PUMA. In the present study, cellular and animal models of SCA3 were used to test the hypothesis that mutant polyglutamine ataxin-3 upregulates Bax expression of cerebellar and pontine nuclei neurons by augmenting transcriptional activity of p53. Electrophoretic mobility shift assay (EMSA) indicated that p53 binding activity to Bax promoter sequence was significantly enhanced in cultured cerebellar neurons expressing mutant ataxin-3-Q79 and pontine nuclei and cerebellum of SCA3 transgenic mice expressing ataxin-3-Q79. The mRNA level of PUMA, a p53-inducible pro-apoptotic gene, was increased in the cerebellum and pontine nuclei of SCA3 transgenic mice and cultured cerebellar neurons expressing ataxin-3-Q79. Mutant polyglutamine ataxin-3 increased the protein level of active phospho-p53(Ser15) in cerebellar and pontine nuclei neurons without affecting mRNA or protein level of p53. Intraperitoneal administration of p53 inhibitor pifithrin-α significantly ameliorated neuronal death in the pontine nuclei of SCA3 transgenic mice. Our results suggest that polyglutamine-expanded ataxin-3 upregulates mRNA expression of Bax and PUMA and causes apoptotic death of affected neurons by enhancing phosphorylation and transcriptional activity of p53., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) play a critical role in regulation of behavioral state. Therefore, elucidation of mechanisms that control their activity is vital for understanding of how switching between wakefulness, sleep and anesthetic states is effectuated. In vivo studies suggest that GABAergic mechanisms within the pons play a critical role in behavioral state switching. However, the postsynaptic, electrophysiological actions of GABA on LDT neurons, as well as the identity of GABA receptors present in the LDT mediating these actions is virtually unexplored. Therefore, we studied the actions of GABA agonists and antagonists on cholinergic LDT cells by performing patch clamp recordings in mouse brain slices. Under conditions where detection of Cl(-) -mediated events was optimized, GABA induced gabazine (GZ)-sensitive inward currents in the majority of LDT neurons. Post-synaptic location of GABA(A) receptors was demonstrated by persistence of muscimol-induced inward currents in TTX and low Ca(2+) solutions. THIP, a selective GABA(A) receptor agonist with a preference for δ-subunit containing GABA(A) receptors, induced inward currents, suggesting the existence of extrasynaptic GABA(A) receptors. LDT cells also possess GABA(B) receptors as baclofen-activated a TTX- and low Ca(2+)-resistant outward current that was attenuated by the GABA(B) antagonists CGP 55845 and saclofen. The tertiapin sensitivity of baclofen-induced outward currents suggests that a G(IRK) mediated this effect. Further, outward currents were never additive with those induced by application of carbachol, suggesting that they were mediated by activation of GABA(B) receptors linked to the same G(IRK) activated in these cells by muscarinic receptor stimulation. Activation of GABA(B) receptors inhibited Ca(2+) increases induced by a depolarizing voltage step shown previously to activate VOCCs in cholinergic LDT neurons. Baclofen-mediated reductions in depolarization-induced Ca(2+) were unaltered by prior emptying of intracellular Ca(2+) stores, but were abolished by low extracellular Ca(2+) and pre-application of nifedipine, indicating that activation of GABA(B) receptors inhibits influx of Ca(2+) involving L-type Ca(2+) channels. Presence of GABA(C) receptors is suggested by the induction of inward current by (E)-4- amino-2-butenoic acid (TACA) and its inhibition by 1,2,5,6-tetrahydropyridine-4-ylmethylphosphinic (TPMPA), a relatively selective agonist and antagonist, respectively, of GABA(C) receptors. All of these GABA-mediated actions were found to occur in histochemically-identified cholinergic neurons. Taken together, these data indicate for the first time that cholinergic neurons of the LDT exhibit functional GABA(A, B and C) receptors, including extrasynaptically located GABA(A) receptors, which may be tonically activated by synaptic overflow of GABA. Accordingly, the activity of cholinergic LDT neurons is likely to be significantly affected by GABAergic tone within the nucleus, and so, demonstrated effects of GABA on behavioral state may be mediated, in part, via direct actions on cholinergic neurons in the LDT., (Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Before hearing onset, the topographic organization of the auditory GABA/glycinergic pathway from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) is refined by synaptic silencing and strengthening. The synaptic mechanisms underlying the developmental strengthening of maintained MNTB-LSO connections are unknown. Here we address this question using whole-cell recordings from LSO neurons in slices prepared from prehearing mice. Minimal and maximal stimulation techniques demonstrated that during the first two postnatal weeks, individual LSO neurons lose about 55% of their initial presynaptic MNTB partners while maintained single-fiber connections become about 14-fold stronger. Analysis of MNTB-evoked miniature events indicates that this strengthening is accompanied by a 2-fold increase in quantal amplitude. Strengthening is not caused by an increase in the probability of release because paired pulse ratios (PPRs) increased from 0.7 in newborn animals to 0.9 around hearing onset, indicating a developmental decrease rather than increase in release probability. In addition, a possible soma-dendritic relocation of MNTB input seems unlikely to underlie their strengthening as indicated by analysis of the rise times of synaptic currents. Taken together, we conclude that the developmental strengthening of MNTB-LSO connections is achieved by a 2-fold increase in quantal size and an 8-fold increase in quantal content., (Copyright © 2010 IBRO. All rights reserved.)

Omnipause neurons (OPNs) are inhibitory neurons located in the midline region of the caudal pons. Their role in gating the discharges of saccade-related burst neurons is well known, but there is no agreement concerning their influence on brainstem neurons that control other muscle groups participating in rapid gaze shifts. In the present study, we inquired whether OPNs project directly to pontobulbar reticulospinal neurons (RSNs) in the cat. Retrograde transport of horseradish peroxidase from the cervical spinal cord was used to label RSNs and an anterograde tracer (biocytin) was iontophoresed at sites of extracellular recording of the OPN activity. Somadendritic characteristics of biocytin-labeled OPNs were largely similar to those obtained previously with intracellular labeling. Three-dimensional reconstruction of axonal trajectories and collaterals revealed that projections of OPNs, regarded as a population, are bilateral. Their terminals were restricted to the reticular formation and midline structures throughout the rostral bulbar and pontine tegmentum. Appositions of synaptic boutons originating from five fully stained OPNs were detected on 38 retrogradely labeled RSNs, each of the OPNs contacting 3-13 cells. The numbers of boutons (1-46; mean 11.8) on the RSN somata and proximal dendrites indicate that the anatomical strength of paired OPN-RSN connections is comparable to that of other similarly studied inhibitory neurons in the cat. The existence of connections with RSNs supports the hypothesis of a generalized influence of OPNs on several effectors participating in orienting gaze shifts as opposed to the idea of their strict specialization for the control of eye saccades., (© 2010 Wiley-Liss, Inc.)

The present study investigated the subnuclear organization of collateralized efferent projection patterns from the mouse parabrachial nucleus (PbN), the second taste relay in rodents, to higher gustatory centers, including the ventroposteromedial nucleus of the thalamus (VPMpc), central nucleus of the amygdala (CeA) and lateral hypothalamus (LH). We made injections of the retrograde tracer red and green latex microspheres into the VMPpc and CeA (VPMpc-CeA group), VMPpc and LH (VPMpc-LH group) or CeA and LH (CeA-LH group, n=6 for each group). Injections into these areas preferentially resulted in retrograde labeling in the ipsilateral PbN in all groups. Cells projecting to the VPMpc, CeA, and LH were generally found in all subnuclei, but were differentially distributed. VPMpc-projecting cells predominated in gustatory-related subnuclei, CeA-projecting neurons predominated in the external lateral (el) subnucleus, and concentrated labeling was observed in the dorsal lateral subnucleus (dl) following LH injection. Double-labeled neurons were found for all groups, almost entirely ipsilaterally and primarily in the medial (m), waist area (wa), ventral lateral (vl) and el subnuclei. These results suggest that PbN neurons in different subdivisions have different projection and collateralization patterns to the VPMpc, CeA and LH. Functional implications of these projections are discussed with an emphasis on their roles in taste., (Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)