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Placebo effects are notable demonstrations of mind-body interactions 1,2 . During pain perception, in the absence of any treatment, an expectation of pain relief can reduce the experience of pain-a phenomenon known as placebo analgesia 3-6 . However, despite the strength of placebo effects and their impact on everyday human experience and the failure of clinical trials for new therapeutics 7 , the neural circuit basis of placebo effects has remained unclear. Here we show that analgesia from the expectation of pain relief is mediated by rostral anterior cingulate cortex (rACC) neurons that project to the pontine nucleus (rACC→Pn)-a precerebellar nucleus with no established function in pain. We created a behavioural assay that generates placebo-like anticipatory pain relief in mice. In vivo calcium imaging of neural activity and electrophysiological recordings in brain slices showed that expectations of pain relief boost the activity of rACC→Pn neurons and potentiate neurotransmission in this pathway. Transcriptomic studies of Pn neurons revealed an abundance of opioid receptors, further suggesting a role in pain modulation. Inhibition of the rACC→Pn pathway disrupted placebo analgesia and decreased pain thresholds, whereas activation elicited analgesia in the absence of placebo conditioning. Finally, Purkinje cells exhibited activity patterns resembling those of rACC→Pn neurons during pain-relief expectation, providing cellular-level evidence for a role of the cerebellum in cognitive pain modulation. These findings open the possibility of targeting this prefrontal cortico-ponto-cerebellar pathway with drugs or neurostimulation to treat pain., (© 2024. The Author(s).)

Survival requires the selection of appropriate behaviour in response to threats, and dysregulated defensive reactions are associated with psychiatric illnesses such as post-traumatic stress and panic disorder 1 . Threat-induced behaviours, including freezing and flight, are controlled by neuronal circuits in the central amygdala (CeA) 2 ; however, the source of neuronal excitation of the CeA that contributes to high-intensity defensive responses is unknown. Here we used a combination of neuroanatomical mapping, in vivo calcium imaging, functional manipulations and electrophysiology to characterize a previously unknown projection from the dorsal peduncular (DP) prefrontal cortex to the CeA. DP-to-CeA neurons are glutamatergic and specifically target the medial CeA, the main amygdalar output nucleus mediating conditioned responses to threat. Using a behavioural paradigm that elicits both conditioned freezing and flight, we found that CeA-projecting DP neurons are activated by high-intensity threats in a context-dependent manner. Functional manipulations revealed that the DP-to-CeA pathway is necessary and sufficient for both avoidance behaviour and flight. Furthermore, we found that DP neurons synapse onto neurons within the medial CeA that project to midbrain flight centres. These results elucidate a non-canonical top-down pathway regulating defensive responses., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The aim of the present study was a refined analysis of neuroinflammation including TMEM119 as a useful microglia-specific marker in forensic assessments of traumatic causes of death, e.g., traumatic brain injury (TBI). Human brain tissue samples were obtained from autopsies and divided into cases with lethal TBI (n = 25) and subdivided into three groups according to their trauma survival time and compared with an age-, gender-, and postmortem interval-matched cohort of sudden cardiovascular fatalities as controls (n = 23). Brain tissue samples next to cortex contusions and surrounding white matter as well as samples of the ipsilateral uninjured brain stem and cerebellum were collected and stained immunohistochemically with antibodies against TMEM119, CD206, and CCR2. We could document the highest number of TMEM119-positive cells in acute TBI death with highly significant differences to the control numbers. CCR2-positive monocytes showed a significantly higher cell count in the cortex samples of TBI cases than in the controls with an increasing number of immunopositive cells over time. The number of CD206-positive M2 microglial cells increased survival time-dependent. After 3 days of survival, the cell number increased significantly in all four regions investigated compared with controls. In sum, we validate a specific and robustly expressed as well as fast reacting microglia marker, TMEM119, which distinguishes microglia from resident and infiltrating macrophages and thus offers a great potential for the estimation of the minimum survival time after TBI.

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.

Pediatric high-grade gliomas are among the deadliest of childhood cancers due to limited knowledge of early driving events in their gliomagenesis and the lack of effective therapies available. In this study, we investigate the oncogenic role of PPM1D, a protein phosphatase often found truncated in pediatric gliomas such as DIPG, and uncover a synthetic lethal interaction between PPM1D mutations and nicotinamide phosphoribosyltransferase (NAMPT) inhibition. Specifically, we show that mutant PPM1D drives hypermethylation of CpG islands throughout the genome and promotes epigenetic silencing of nicotinic acid phosphoribosyltransferase (NAPRT), a key gene involved in NAD biosynthesis. Notably, PPM1D mutant cells are shown to be sensitive to NAMPT inhibitors in vitro and in vivo, within both engineered isogenic astrocytes and primary patient-derived model systems, suggesting the possible application of NAMPT inhibitors for the treatment of pediatric gliomas. Overall, our results reveal a promising approach for the targeting of PPM1D mutant tumors, and define a critical link between oncogenic driver mutations and NAD metabolism, which can be exploited for tumor-specific cell killing.

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.

The cerebellum is involved in the control of movement, emotional responses, and reward processing. The tree shrew is the closest living relative of primates. However, little is known not only about the systematic nomenclature for the tree shrew cerebellum but also about the detailed neurochemical characterization and afferent projections. In this study, Nissl staining and acetylcholinesterase histochemistry were used to reveal anatomical features of the cerebellum of tree shrews (Tupaia belangeri chinensis). The cerebellar cortex presented a laminar structure. The morphological characteristics of the cerebellum were comprehensively described in the coronal, sagittal, and horizontal sections. Moreover, distributive maps of calbindin-immunoreactive (-ir) cells in the Purkinje cell layer of the cerebellum of tree shrews were depicted using coronal, sagittal, and horizontal schematics. In addition, 5th cerebellar lobule (5Cb)-projecting neurons were present in the pontine nuclei, reticular nucleus, spinal vestibular nucleus, ventral spinocerebellar tract, and inferior olive of the tree shrew brain. The anterior part of the paramedian lobule of the cerebellum (PMa) received mainly strong innervation from the lateral reticular nucleus, inferior olive, pontine reticular nucleus, spinal trigeminal nucleus, pontine nuclei, and reticulotegmental nucleus of the pons. The present results provide the first systematic nomenclature, detailed atlas of the whole cerebellum, and whole-brain mapping of afferent projections to the 5Cb and PMa in tree shrews. Our findings provide morphological support for tree shrews as an alternative model for studies of human cerebellar pathologies., (© 2018 Wiley Periodicals, Inc.)

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.

Inputs from the ventral hippocampus (vHPC) to the prefrontal cortex (PFC) play a key role in working memory and emotional control. However, little is known about how excitatory inputs from the vHPC engage different populations of neurons in the PFC. Here we use optogenetics and whole-cell recordings to study the cell-type specificity of synaptic connections in acute slices from the mouse PFC. We first show that vHPC inputs target pyramidal neurons whose cell bodies are located in layer (L)2/3 and L5 of infralimbic (IL) PFC, but only in L5 of prelimbic (PL) PFC, and not L6 of either IL or PL. We then compare connections onto different classes of projection neurons located in these layers and subregions of PFC. We establish vHPC inputs similarly contact corticocortical (CC) and cortico-amygdala neurons in L2/3 of IL, but preferentially target CC neurons over cortico-pontine neurons in L5 of both IL and PL. Of all these neurons, we determine that vHPC inputs are most effective at driving action potential (AP) firing of CC neurons in L5 of IL. We also show this connection exhibits frequency-dependent facilitation, with repetitive activity enhancing AP firing of IL L5 CC neurons, even in the presence of feedforward inhibition. Our findings reveal how vHPC inputs engage defined populations of projection neurons in the PFC, allowing preferentially activation of the intratelencephalic network. SIGNIFICANCE STATEMENT We examined the impact of connections from the ventral hippocampus (vHPC) onto different projection neurons in the mouse prefrontal cortex (PFC). We found vHPC inputs were strongest at corticocortical neurons in layer 5 of infralimbic PFC, where they robustly evoked action potential firing, including during repetitive activity with intact feedforward inhibition., (Copyright © 2018 the authors 0270-6474/18/387351-13$15.00/0.)

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.)

During brainstem development, newborn neurons originating from the rhombic lip embark on exceptionally long migrations to generate nuclei important for audition, movement, and respiration. Along the way, this highly motile population passes several cranial nerves yet remains confined to the CNS. We found that Ntn1 accumulates beneath the pial surface separating the CNS from the PNS, with gaps at nerve entry sites. In mice null for Ntn1 or its receptor DCC, hindbrain neurons enter cranial nerves and migrate into the periphery. CNS neurons also escape when Ntn1 is selectively lost from the sub-pial region (SPR), and conversely, expression of Ntn1 throughout the mutant hindbrain can prevent their departure. These findings identify a permissive role for Ntn1 in maintaining the CNS-PNS boundary. We propose that Ntn1 confines rhombic lip-derived neurons by providing a preferred substrate for tangentially migrating neurons in the SPR, preventing their entry into nerve roots., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Objective: We investigated the neural pathway for tear secretion from the lacrimal gland of New Zealand White rabbits., Methods: Nine healthy adult New Zealand White rabbits were randomly divided into three experimental groups, namely, an irritant-stimulated group, a non-stimulated group, and a saline-stimulated group. Sanitized dry cotton swabs with menthol were used to wipe both of the rabbits' eyelids in the irritant-stimulated group, and the non-stimulated group and saline- stimulated group were compared as controls. The animals in the three groups were killed 2h later and the expressions of c-Fos in the frontal cortex, hippocampus, hypothalamus, pons, and medulla oblongata of the rabbits were detected using immunofluorescence labeling. According to the distribution of c-Fos protein expression, 12 healthy adult New Zealand rabbits were similarly divided into three groups for retrograde tract tracing via pseudorabies virus (PRV) injection into the lacrimal gland. Immunofluorescence labeling was used to analyze PRV-infected neurons in the brains of rabbits after survival for 30h, 38h, and 46h., Results: The most c-Fos-positive immunolabeled cells were observed in the menthol-stimulated group, whereas fewer c-Fos-positive immunolabeled cells were observed in the saline-stimulated group.The non-treated group showed the least c-Fos-positive immunolabeled cells. At 30h after PRV injection, PRV-positive neurons were found only in the superior salivary nucleus of the pons (SSN). At 38h, PRV-infected neurons were observed in the lateral nucleus of the superior olive (LSO) and the medial nucleus of the superior olive (MSO). At 46h, PRV-infected neurons were found in the nucleus of the trapezoid body (Tz) and the hypothalamic paraventricular nucleus (PVN), and their distributions were dense in the LSO and MSO., Conclusions: Menthol-induced c-Fos protein expression and PRV-mediated tract tracing suggest that in New Zealand White rabbits, the neural pathway that regulates tear secretion from the lacrimal gland proceeds from the PVN to the superior olivary complex of the pons to the SSN and finally to the lacrimal gland., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: Fos protein expression in catecholamine-synthesizing neurons of the substantia nigra (SN) pars compacta (SNC, A8), pars reticulata (SNR, A9), and pars lateralis (SNL), the ventral tegmental area (VTA, A10), the locus coeruleus (LC, A6) and subcoeruleus (sLC), the ventrolateral pons (PON-A5), the nucleus of the solitary tract (NTS-A2), the area postrema (AP), and the ventrolateral medulla (VLM-A1) was quantitatively evaluated aft er a single administration of asenapine (ASE) (designated for schizophrenia treatment) in male Wistar rats preconditioned with a chronic unpredictable variable mild stress (CMS) for 21 days. Th e aim of the present study was to reveal whether a single ASE treatment may 1) activate Fos expression in the brain areas selected; 2) activate tyrosine hydroxylase (TH)-synthesizing cells displaying Fos presence; and 3) be modulated by CMS preconditioning., Methods: Control (CON), ASE, CMS, and CMS+ASE groups were used. CMS included restraint, social isolation, crowding, swimming, and cold. Th e ASE and CMS+ASE groups received a single dose of ASE (0.3 mg/kg, s.c.) and CON and CMS saline (300 μl/rat, s.c.). The animals were sacrificed 90 min aft er the treatments. Fos protein and TH-labeled immunoreactive perikarya were analyzed on double labeled histological sections and enumerated on captured pictures using combined light and fluorescence microscope illumination., Results: Saline or CMS alone did not promote Fos expression in any of the structures investigated. ASE alone or in combination with CMS elicited Fos expression in two parts of the SN (SNC, SNR) and the VTA. Aside from some cells in the central gray tegmental nuclei adjacent to LC, where a small number of Fos profiles occurred, none or negligible Fos occurrence was detected in the other structures investigated including the LC and sLC, PON-A5, NTS-A2, AP, and VLM-A1. CMS preconditioning did not infl uence the level of Fos induction in the SN and VTA elicited by ASE administration. Similarly, the ratio between the amount of free Fos and Fos colocalized with TH was not aff ected by stress preconditioning in the SNC, SNR, and the VTA., Conclusions: Th e present study provides an anatomical/functional knowledge about the nature of the acute ASE treatment on the catecholamine-synthesizing neurons activity in certain brain structures and their missing interplay with the CMS preconditioning.

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.)

Study Objectives: Coma and chronic sleepiness are common after traumatic brain injury (TBI). Here, we explored whether injury to arousal-promoting brainstem neurons occurs in patients with fatal TBI., Methods: Postmortem examination of 8 TBI patients and 10 controls., Results: Compared to controls, TBI patients had 17% fewer serotonergic neurons in the dorsal raphe nucleus (effect size: 1.25), but the number of serotonergic neurons did not differ in the median raphe nucleus. TBI patients also had 29% fewer noradrenergic neurons in the locus coeruleus (effect size: 0.96). The number of cholinergic neurons in the pedunculopontine and laterodorsal tegmental nuclei (PPT/LDT) was similar in TBI patients and controls., Conclusions: TBI injures arousal-promoting neurons of the mesopontine tegmentum, but this injury is less severe than previously observed in hypothalamic arousal-promoting neurons. Most likely, posttraumatic arousal disturbances are not primarily caused by damage to these brainstem neurons, but arise from an aggregate of injuries, including damage to hypothalamic arousal nuclei and disruption of other arousal-related circuitries., (© 2016 Associated Professional Sleep Societies, LLC.)

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.

We recently characterized physiologically a pontine reticulospinal (pRS) projection in the neonatal mouse that mediates synaptic effects on spinal motoneurons via parallel uncrossed and crossed pathways (Sivertsen et al. [2014] J Neurophysiol 112:1628-1643). Here we characterize the origins, anatomical organization, and supraspinal axon trajectories of these pathways via retrograde tracing from the high cervical spinal cord. The two pathways derive from segregated populations of ipsilaterally and contralaterally projecting pRS neurons with characteristic locations within the pontine reticular formation (PRF). We obtained estimates of relative neuron numbers by counting from sections, digitally generated neuron position maps, and 3D reconstructions. Ipsilateral pRS neurons outnumber contralateral pRS neurons by threefold and are distributed about equally in rostral and caudal regions of the PRF, whereas contralateral pRS neurons are concentrated in the rostral PRF. Ipsilateral pRS neuron somata are on average larger than contralateral. No pRS neurons are positive in transgenic mice that report the expression of GAD, suggesting that they are predominantly excitatory. Putative GABAergic interneurons are interspersed among the pRS neurons, however. Ipsilateral and contralateral pRS axons have distinctly different trajectories within the brainstem. Their initial spinal funicular trajectories also differ, with ipsilateral and contralateral pRS axons more highly concentrated medially and laterally, respectively. The larger size and greater number of ipsilateral vs. contralateral pRS neurons is compatible with our previous finding that the uncrossed projection transmits more reliably to spinal motoneurons. The information about supraspinal and initial spinal pRS axon trajectories should facilitate future physiological assessment of synaptic connections between pRS neurons and spinal neurons., (© 2015 Wiley Periodicals, Inc.)

Mammalian sleep comprises rapid eye movement (REM) sleep and non-REM (NREM) sleep. To functionally isolate from the complex mixture of neurons populating the brainstem pons those involved in switching between REM and NREM sleep, we chemogenetically manipulated neurons of a specific embryonic cell lineage in mice. We identified excitatory glutamatergic neurons that inhibit REM sleep and promote NREM sleep. These neurons shared a common developmental origin with neurons promoting wakefulness; both derived from a pool of proneural hindbrain cells expressing Atoh1 at embryonic day 10.5. We also identified inhibitory γ-aminobutyric acid-releasing neurons that act downstream to inhibit REM sleep. Artificial reduction or prolongation of REM sleep in turn affected slow-wave activity during subsequent NREM sleep, implicating REM sleep in the regulation of NREM sleep., (Copyright © 2015, American Association for the Advancement of Science.)

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.

Rapid eye movement (REM) sleep is a distinct brain state characterized by activated electroencephalogram and complete skeletal muscle paralysis, and is associated with vivid dreams. Transection studies by Jouvet first demonstrated that the brainstem is both necessary and sufficient for REM sleep generation, and the neural circuits in the pons have since been studied extensively. The medulla also contains neurons that are active during REM sleep, but whether they play a causal role in REM sleep generation remains unclear. Here we show that a GABAergic (γ-aminobutyric-acid-releasing) pathway originating from the ventral medulla powerfully promotes REM sleep in mice. Optogenetic activation of ventral medulla GABAergic neurons rapidly and reliably initiated REM sleep episodes and prolonged their durations, whereas inactivating these neurons had the opposite effects. Optrode recordings from channelrhodopsin-2-tagged ventral medulla GABAergic neurons showed that they were most active during REM sleep (REMmax), and during wakefulness they were preferentially active during eating and grooming. Furthermore, dual retrograde tracing showed that the rostral projections to the pons and midbrain and caudal projections to the spinal cord originate from separate ventral medulla neuron populations. Activating the rostral GABAergic projections was sufficient for both the induction and maintenance of REM sleep, which are probably mediated in part by inhibition of REM-suppressing GABAergic neurons in the ventrolateral periaqueductal grey. These results identify a key component of the pontomedullary network controlling REM sleep. The capability to induce REM sleep on command may offer a powerful tool for investigating its functions.

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.)

Sensorimotor integration is crucial to perception and motor control. How and where this process takes place in the brain is still largely unknown. Here we analyze the cerebellar contribution to sensorimotor integration in the whisker system of mice. We identify an area in the cerebellum where cortical sensory and motor inputs converge at the cellular level. Optogenetic stimulation of this area affects thalamic and motor cortex activity, alters parameters of ongoing movements and thereby modifies qualitatively and quantitatively touch events against surrounding objects. These results shed light on the cerebellum as an active component of sensorimotor circuits and show the importance of sensorimotor cortico-cerebellar loops in the fine control of voluntary movements.

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.

Appetite suppression occurs after a meal and in conditions when it is unfavourable to eat, such as during illness or exposure to toxins. A brain region proposed to play a role in appetite suppression is the parabrachial nucleus, a heterogeneous population of neurons surrounding the superior cerebellar peduncle in the brainstem. The parabrachial nucleus is thought to mediate the suppression of appetite induced by the anorectic hormones amylin and cholecystokinin, as well as by lithium chloride and lipopolysaccharide, compounds that mimic the effects of toxic foods and bacterial infections, respectively. Hyperactivity of the parabrachial nucleus is also thought to cause starvation after ablation of orexigenic agouti-related peptide neurons in adult mice. However, the identities of neurons in the parabrachial nucleus that regulate feeding are unknown, as are the functionally relevant downstream projections. Here we identify calcitonin gene-related peptide-expressing neurons in the outer external lateral subdivision of the parabrachial nucleus that project to the laterocapsular division of the central nucleus of the amygdala as forming a functionally important circuit for suppressing appetite. Using genetically encoded anatomical, optogenetic and pharmacogenetic tools, we demonstrate that activation of these neurons projecting to the central nucleus of the amygdala suppresses appetite. In contrast, inhibition of these neurons increases food intake in circumstances when mice do not normally eat and prevents starvation in adult mice whose agouti-related peptide neurons are ablated. Taken together, our data demonstrate that this neural circuit from the parabrachial nucleus to the central nucleus of the amygdala mediates appetite suppression in conditions when it is unfavourable to eat. This neural circuit may provide targets for therapeutic intervention to overcome or promote appetite.

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.

Stimulation of neurons in the cuneiform nucleus (CnF) produces antinociception and cardiovascular responses that could be mediated, in part, by noradrenergic neurons that innervate the spinal cord dorsal horn. The present study determined the projections of neurons in the CnF to the pontine noradrenergic neurons in the A5, A6 (locus coeruleus), and A7 cell groups that are known to project to the spinal cord. Injections of the anterograde tracer, biotinylated dextran amine in the CnF of Sasco Sprague-Dawley rats labeled axons located near noradrenergic neurons that were visualized by processing tissue sections for tyrosine hydroxylase-immunoreactivity. Anterogradely labeled axons were more dense on the side ipsilateral to the BDA deposit. Both A7 and A5 cell groups received dense projections from neurons in the CnF, whereas locus coeruleus received only a sparse projection. Highly varicose anterogradely labeled axons from the CnF were found in close apposition to dendrites and somata of tyrosine hydroxylase-immunoreactive neurons in pontine tegmentum. Although definitive evidence for direct pathways from CnF neurons to the pontine noradrenergic cell groups requires ultrastructural analysis, the results of the present studies provide presumptive evidence of direct projections from neurons in the CnF to the pontine noradrenergic neurons of the A7, locus coeruleus, and A5 cell groups. These results support the suggestion that the analgesia and cardiovascular responses produced by stimulation of neurons in the CnF may be mediated, in part, by pontine noradrenergic neurons., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The steep calcium dependence of synaptic strength that has been observed at many synapses is thought to reflect a calcium dependence of the probability of vesicular exocytosis (p), with the cooperativity of three to six corresponding to the multiple calcium ion binding sites on the calcium sensor responsible for exocytosis. Here we test the hypothesis that the calcium dependence of the effective size of the readily releasable pool (RRP) also contributes to the calcium dependence of release at the calyx of Held synapse in mice. Using two established methods of quantifying neurotransmitter release evoked by action potentials (effective RRP), we find that when calcium influx is changed by altering the external calcium concentration, the calcium cooperativity of p is insufficient to account for the full calcium dependence of EPSC size; the calcium dependence of the RRP size also contributes. Reducing calcium influx by blocking R-type voltage-gated calcium channels (VGCCs) with Ni(2+), or by blocking P/Q-type VGCCs with ω-agatoxin IVA also changes EPSC amplitude by reducing both p and the effective RRP size. This suggests that the effective RRP size is dependent on calcium influx through VGCCs. Furthermore, activation of GABAB receptors, which reduces presynaptic calcium through VGCCs without other significant effects on release, also reduces the effective RRP size in addition to reducing p. These findings indicate that calcium influx regulates the RRP size along with p, which contributes to the calcium dependence of synaptic strength, and it influences the manner in which presynaptic modulation of presynaptic calcium channels affects neurotransmitter release.

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.

Determining how long-range synaptic inputs engage pyramidal neurons in primary motor cortex (M1) is important for understanding circuit mechanisms involved in regulating movement. We used channelrhodopsin-2-assisted circuit mapping to characterize the long-range excitatory synaptic connections made by multiple cortical and thalamic areas onto pyramidal neurons in mouse vibrissal motor cortex (vM1). Each projection innervated vM1 pyramidal neurons with a unique laminar profile. Collectively, the profiles for different sources of input partially overlapped and spanned all cortical layers. Specifically, orbital cortex (OC) inputs primarily targeted neurons in L6. Secondary motor cortex (M2) inputs excited neurons mainly in L5B, including pyramidal tract neurons. In contrast, thalamocortical inputs from anterior motor-related thalamic regions, including VA/VL (ventral anterior thalamic nucleus/ventrolateral thalamic nucleus), targeted neurons in L2/3 through L5B, but avoided L6. Inputs from posterior sensory-related thalamic areas, including POm (posterior thalamic nuclear group), targeted neurons only in the upper layers (L2/3 and L5A), similar to inputs from somatosensory (barrel) cortex. Our results show that long-range excitatory inputs target vM1 pyramidal neurons in a layer-specific manner. Inputs from sensory-related cortical and thalamic areas preferentially target the upper-layer pyramidal neurons in vM1. In contrast, inputs from OC and M2, areas associated with volitional and cognitive aspects of movements, bypass local circuitry and have direct monosynaptic access to neurons projecting to brainstem and thalamus.

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.)

Ca(V)2.1 Ca(2+) channels have a dominant and specific role in initiating fast synaptic transmission at central excitatory synapses, through a close association between release sites and calcium sensors. Familial hemiplegic migraine type 1 (FHM-1) is an autosomal-dominant subtype of migraine with aura, caused by missense mutations in the CACNA1A gene that encodes the α(1A) pore-forming subunit of Ca(V)2.1 channel. We used knock-in (KI) transgenic mice harboring the FHM-1 mutation R192Q to study the consequences of this mutation in neurotransmission at the giant synapse of the auditory system formed by the presynaptic calyx of Held terminal and the postsynaptic neurons of the medial nucleus of the trapezoid body (MNTB). Although synaptic transmission seems unaffected by low-frequency stimulation in physiological Ca(2+) concentration, we observed that with low Ca(2+) concentrations (<1 mM) excitatory postsynaptic currents (EPSCs) showed increased amplitudes in R192Q KI mice compared with wild type (WT), meaning significant differences in the nonlinear calcium dependence of nerve-evoked transmitter release. In addition, when EPSCs were evoked by broadened presynaptic action potentials (achieved by inhibition of K(+) channels) via Ca(v)2.1-triggered exocytosis, R192Q KI mice exhibited further enhancement of EPSC amplitude and charge compared with WT mice. Repetitive stimulation of afferent axons to the MNTB at different frequencies caused short-term depression of EPSCs that recovered significantly faster in R192Q KI mice than in WT mice. Faster recovery in R192Q KI mice was prevented by the calcium chelator EGTA-AM, pointing to enlarged residual calcium as a key factor in accelerating the replenishment of synaptic vesicles.

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.)