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Neurons in the nucleus raphe interpositus have tonic activity that suppresses saccadic burst neurons (BNs) during eye fixations, and that is inhibited before and during saccades in all directions (omnipause neurons, OPNs). We have previously demonstrated via intracellular recording and anatomical staining in anesthetized cats of both sexes that OPNs are inhibited by BNs in the medullary reticular formation (horizontal inhibitory BNs, IBNs). These horizontal IBNs receive monosynaptic input from the caudal horizontal saccade area of the superior colliculus (SC), and then produce monosynaptic inhibition in OPNs, providing a mechanism to trigger saccades. However, it is well known that the neural circuits driving horizontal components of saccades are independent from the circuits driving vertical components. Thus, our previous results are unable to explain how purely vertical saccades are triggered. Here, we again apply intracellular recording to show that a disynaptic vertical IBN circuit exists, analogous to the horizontal circuit. Specifically, we show that stimulation of the SC rostral vertical saccade area produces disynaptic inhibition in OPNs, which is not abolished by midline section between the horizontal IBNs. This excludes the possibility that horizontal IBNs could be responsible for the OPN inhibition during vertical saccades. We then show that vertical IBNs in the interstitial nucleus of Cajal, which receive monosynaptic input from rostral SC, are responsible for the disynaptic inhibition of OPNs. These results indicate that a similarly functioning SC-IBN-OPN circuit exists for both the horizontal and vertical oculomotor pathways. These two IBN-mediated circuits are capable of triggering saccades in any direction. [ABSTRACT FROM AUTHOR]

Omnipause neurons (OPNs) in the nucleus raphe interpositus have tonic activity while the eyes are stationary ("fixation") but stop firing immediately before and during saccades. To locate the source of suppression, we analyzed synaptic inputs from the rostral and caudal superior colliculi (SCs) to OPNs by using intracellular recording and staining, and investigated pathways transmitting the inputs in anesthetized cats of both sexes. Electrophysiologically or morphologically identified OPNs received monosynaptic excitation from the rostral SCs with contralateral dominance, and received disynaptic inhibition from the caudal SCs with ipsilateral dominance. Cutting the tectoreticular tract transversely between the contralateral OPN and inhibitory burst neuron (IBN) regions eliminated inhibition from the caudal SCs, but not excitation from the rostral SCs in OPNs. In contrast, a midline section between IBN regions eliminated disynaptic inhibition in OPNs from the caudal SCs but did not affect the monosynaptic excitation from the rostral SCs. Stimulation of the contralateral IBN region evoked monosynaptic inhibition in OPNs, which was facilitated by preconditioning SC stimulation. Three-dimensional reconstruction of HRP-stained cells revealed that individual OPNs have axons that terminate in the opposite IBN area, while individual IBNs have axon collaterals to the opposite OPN area. These results show that there are differences in the neural circuit from the rostral and caudal SCs to the brainstem premotor circuitry and that IBNs suppress OPNs immediately before and during saccades. Thus, the IBNs, which are activated by caudal SC saccade neurons, shut down OPN firing and help to trigger saccades and suppress ("latch") OPN activity during saccades. SIGNIFICANCE STATEMENT Saccades are the fastest eye movements to redirect gaze to an object of interest and bring its image on the fovea for fixation. Burst neurons (BNs) and omnipause neurons (OPNs) which behave reciprocally in the brainstem, are important for saccade generation and fixation. This study investigated unsolved important questions about where these neurons receive command signals and how they interact for initiating saccades from visual fixation. The results show that the rostral superior colliculi (SCs) excite OPNs monosynaptically for fixation, whereas the caudal SCs monosynaptically excite inhibitory BNs, which then directly inhibit OPNs for the initiation of saccades. This inhibition from the caudal SCs may account for the omnipause behavior of OPNs for initiation and maintenance of saccades in all directions., (Copyright © 2022 the authors.)

Ca 2+ -dependent activator protein for secretion 2 (CAPS2) regulates dense-core vesicle (DCV) exocytosis to facilitate peptidergic and catecholaminergic transmitter release. CAPS2 deficiency in mice has mild neuronal effects but markedly impairs social behavior. Rare de novo Caps2 alterations also occur in autism spectrum disorder, although whether CAPS2-mediated release influences social behavior remains unclear. Here, we demonstrate that CAPS2 is associated with DCV exocytosis-mediated release of the social interaction modulatory peptide oxytocin (OXT). CAPS2 is expressed in hypothalamic OXT neurons and localizes to OXT nerve projection and OXT release sites, such as the pituitary. Caps2 KO mice exhibited reduced plasma albeit increased hypothalamic and pituitary OXT levels, indicating insufficient release. OXT neuron-specific Caps2 conditional KO supported CAPS2 function in pituitary OXT release, also affording impaired social interaction and recognition behavior that could be ameliorated by exogenous OXT administered intranasally. Thus, CAPS2 appears critical for OXT release, thereby being associated with social behavior. SIGNIFICANCE STATEMENT The role of the neuropeptide oxytocin in enhancing social interaction and social bonding behavior has attracted considerable public and neuroscientific attention. A central issue in oxytocin biology concerns how oxytocin release is regulated. Our study provides an important insight into the understanding of oxytocin-dependent social behavior from the perspective of the CAPS2-regulated release mechanism., (Copyright © 2021 the authors.)

ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4 +/- /ARF5 -/- mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4 +/- /ARF5 -/- mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4 +/- /ARF5 -/- mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of pore-forming voltage-dependent Na + channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4 +/- /ARF5 -/- mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4 +/- /ARF5 -/- mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS. SIGNIFICANCE STATEMENT We found that decreasing the expression of class II ARF proteins, through the generation of ARF4 +/- /ARF5 -/- mice, impairs Nav1.6 distribution to the axon initial segment (AIS) of cerebellar Purkinje cells (PCs), thereby resulting in the impairment of action potential firing of PCs. The ARF4 +/- /ARF5 -/- mutant mice exhibited movement-associated essential tremor (ET)-like behavior with pharmacological profiles similar to those in ET patients. The exogenous expression of ARF5 reduced the tremor phenotype and restored the localization of Nav1.6 immunoreactivity to the AIS in ARF4 +/- /ARF5 -/- mice. Thus, our results suggest that class II ARFs are involved in the localization of Nav1.6 to the AISs in cerebellar PCs and that the reduction of class II ARF activity leads to ET-like movement disorder., (Copyright © 2019 the authors.)

Ca(2+)-dependent activator protein for secretion 1 (CAPS1) plays a regulatory role in the dense-core vesicle (DCV) exocytosis pathway, but its functions at the cellular and synaptic levels in the brain are essentially unknown because of neonatal death soon after birth in Caps1 knock-out mice. To clarify the functions of the protein in the brain, we generated two conditional knock-out (cKO) mouse lines: 1) one lacking Caps1 in the forebrain; and 2) the other lacking Caps1 in the cerebellum. Both cKO mouse lines were born normally and grew to adulthood, although they showed subcellular and synaptic abnormalities. Forebrain-specific Caps1 cKO mice showed reduced immunoreactivity for the DCV marker secretogranin II (SgII) and the trans-Golgi network (TGN) marker syntaxin 6, a reduced number of presynaptic DCVs, and dilated trans-Golgi cisternae in the CA3 region. Cerebellum-specific Caps1 cKO mice had decreased immunoreactivity for SgII and brain-derived neurotrophic factor (BDNF) along the climbing fibers. At climbing fiber-Purkinje cell synapses, the number of DCVs was markedly lower and the number of synaptic vesicles was also reduced. Correspondingly, the mean amplitude of EPSCs was decreased, whereas paired-pulse depression was significantly increased. Our results suggest that loss of CAPS1 disrupts the TGN-DCV pathway, which possibly impairs synaptic transmission by reducing the presynaptic release probability.

Cerebellar climbing fibers (CFs) provide powerful excitatory input to Purkinje cells (PCs), which represent the sole output of the cerebellar cortex. Recent discoveries suggest that CFs have information-rich signaling properties important for cerebellar function, beyond eliciting the well known all-or-none PC complex spike. CF morphology has not been quantitatively analyzed at the same level of detail as its biophysical properties. Because morphology can greatly influence function, including the capacity for information processing, it is important to understand CF branching structure in detail, as well as its variability across and within arbors. We have digitally reconstructed 68 rat CFs labeled using biotinylated dextran amine injected into the inferior olive and comprehensively quantified their morphology. CF structure was considerably diverse even within the same anatomical regions. Distinctly identifiable primary, tendril, and distal branches could be operationally differentiated by the relative size of the subtrees at their initial bifurcations. Additionally, primary branches were more directed toward the cortical surface and had fewer and less pronounced synaptic boutons, suggesting they prioritize efficient and reliable signal propagation. Tendril and distal branches were spatially segregated and bouton dense, indicating specialization in signal transmission. Furthermore, CFs systematically targeted molecular layer interneuron cell bodies, especially at terminal boutons, potentially instantiating feedforward inhibition on PCs. This study offers the most detailed and comprehensive characterization of CF morphology to date. The reconstruction files and metadata are publicly distributed at NeuroMorpho.org.

The olivocerebellar climbing fiber projection pattern is closely correlated with the pattern of aldolase C expression in cerebellar Purkinje cells. Based on this expression pattern, the olivocerebellar projection can be classified into five "groups" of functional compartments. Each group originates from a subarea within the inferior olive that projects to multiple cortical stripes of Purkinje cells, all of which are either aldolase C positive or aldolase C negative. However, no equivalent compartmental organization has been demonstrated in the cerebellar nuclei (CN). Thus, in the CN of the rat, we systematically mapped the location of olivonuclear projections belonging to the five groups and determined their relationship to the expression of aldolase C in Purkinje cell axonal terminals. The CN were divided into caudoventral aldolase C-positive and rostrodorsal aldolase C-negative parts. The olivonuclear terminations from the five groups projected topographically to five separate compartments within the CN that partly crossed the traditional boundaries that define the fastigial, interposed, and dentate nuclei. Each compartment had mostly uniform cytoarchitecture and the same aldolase C expression (either positive or negative) that was found in the corresponding olivocortical projection. These results suggest a new view of the organization of the CN whereby the pattern of olivonuclear terminations links portions of different CN together. We propose that each compartment in the CN, along with its corresponding olivary subarea and cortical stripes, may be related to a different aspect of motor control.

Aldolase C (zebrin) expression in Purkinje cells reveals stripe-shaped compartments in the cerebellar cortex. However, it is not clear how these compartments are related to cerebellar functional localization. Therefore, we identified olivocerebellar projections to aldolase C compartments by labeling climbing fibers with biotinylated dextran injected into various small areas within the inferior olive in rats. Specific rostral and caudal aldolase C compartments were linked in an orderly manner by common olivocerebellar projection across the rostrocaudal boundary on lobule VIc-crus Ib. Based on the localization of the olivary origins of projection to similar compartments, the compartments and olivocerebellar projections could be sorted into five groups: group I, positive compartments extending from the posterior lobe to the anterior lobe innervated by the principal olive and some neighboring areas; group II, positive compartments localized within the posterior lobe innervated by several medial subnuclei; group III, vermal and central negative compartments innervated by the centrocaudal medial accessory olive; group IV, negative and lightly positive compartments in the hemisphere and the rostral and caudal pars intermedia innervated by the dorsal accessory olive and some neighboring areas; group V, the flocculus and nodulus. The olivocerebellar topography within each group was simple and suggests an "orientation axis" within the concerned parts of the inferior olive. Furthermore, parts of the inferior olive in each group receive specific afferent inputs, indicating a close relationship between aldolase C compartments and functional localization. Thus, the five-group scheme we propose here may integrate the molecular, topographic, and functional organization of the cerebellum.

The functional partitioning of the cerebellar cortex depends on the projection patterns of its afferent and efferent neurons. However, the entire morphology of individual projection neurons has been demonstrated in only a few classes of neurons in the vertebrate CNS. To investigate the contribution of the projection pattern of individual olivocerebellar axons to the cerebellar functional compartmentalization, we labeled individual olivocerebellar axons, which terminate in the cerebellar cortex as climbing fibers, with biotinylated dextran amine injected into the inferior olive in the rat, and completely reconstructed the entire trajectories of 34 olivocerebellar axons from serial sections of the cerebellum and medulla. Single axons had seven climbing fibers on average, which terminated at similar distances from the midline in a single or in multiple lobules. Cortical projection areas of adjacent olivary neurons were clustered as narrow but separate longitudinal segments and often innervated by collaterals of single neurons. Comparison of the cerebellar distribution of olivocerebellar axons arising from different sites within a single olivary subnucleus indicated that slightly distant neurons projected to complementary sets of such segments in a single longitudinal band. Several of these longitudinal bands formed a so-called parasagittal zone innervated by a subnucleus of the inferior olive. Single olivocerebellar axons projected rostrocaudally to segments within a single band but did not project mediolaterally to multiple bands. These results suggest fine substructural organization in the cerebellar compartmentalization that may represent functional units.