Rubrocerebellar Feedback Loop Isolates the Interposed Nucleus as an Independent Processor of Corollary Discharge Information in Mice.

Understanding cerebellar contributions to motor coordination requires deeper insight into how the output structures of the cerebellum, the cerebellar nuclei, integrate their inputs and influence downstream motor pathways. The magnocellular red nucleus (RNm), a brainstem premotor structure, is a major target of the interposed nucleus (IN), and has also been described in previous studies to send feedback collaterals to the cerebellum. Because such a pathway is in a key position to provide motor efferent information to the cerebellum, satisfying predictions about the use of corollary discharge in cerebellar computations, we studied it in mice of both sexes. Using anterograde viral tracing, we show that innervation of cerebellum by rubrospinal neuron collaterals is remarkably selective for the IN compared with the cerebellar cortex. Optogenetic activation of the pathway in acute mouse brain slices drove IN activity despite small amplitude synaptic currents, suggesting an active role in IN information processing. Monosynaptic transsynaptic rabies tracing indicated the pathway contacts multiple cell types within the IN. By contrast, IN inputs to the RNm targeted a region that lacked inhibitory neurons. Optogenetic drive of IN inputs to the RNm revealed strong, direct excitation but no inhibition of RNm neurons. Together, these data indicate that the cerebellar nuclei are under afferent control independent of the cerebellar cortex, potentially diversifying its roles in motor control.SIGNIFICANCE STATEMENT The common assumption that all cerebellar mossy fibers uniformly collateralize to the cerebellar nuclei and cortex underlies classic models of convergent Purkinje influence on cerebellar output. Specifically, mossy fibers are thought to both directly excite nuclear neurons and drive polysynaptic feedforward inhibition via Purkinje neurons, setting up a fundamental computational unit. Here we present data that challenge this rule. A dedicated cerebellar nuclear afferent comprised of feedback collaterals from premotor rubrospinal neurons can directly modulate IN output independent of Purkinje cell modulation. In contrast to the IN-RNm pathway, the RNm-IN feedback pathway targets multiple cell types, potentially influencing both motor output pathways and nucleo-olivary feedback.

Cerebellar loops: a review of the nucleocortical pathway.

Feedback pathways are a common circuit motif in vertebrate brains. Reciprocal interconnectivity is seen between the cerebral cortex and thalamus as well as between basal ganglia structures, for example. Here, we review the literature on the nucleocortical pathway, a feedback pathway from the cerebellar nuclei to the cerebellar cortex, which has been studied anatomically but has remained somewhat obscure. This review covers the work examining this pathway on a number of levels, ranging from its existence in numerous species, its organization within cerebellar circuits, its cellular composition, and a discussion of its potential roles in motor control. Recent interest in cerebellar modular organization raises the profile of this neglected cerebellar pathway, and it is hoped that this review will consolidate knowledge gained over several decades of research into a useful format, spurring new investigations into this evolutionarily conserved pathway.

Analysis of N- and O-Linked Glycosylation: Differential Glycosylation after Rat Spinal Cord Injury.

Glycosylation is a fundamental cellular process that has a dramatic impact on the functionality of glycoconjugates such as proteins or lipids and mediates many different biological interactions including cell migration, cellular signaling, and synaptic interactions in the nervous system. In spinal cord injury (SCI), all of these cellular processes are altered, but the potential contributions of glycosylation changes to these alterations has not been thoroughly investigated. We studied the glycosylation of injured spinal cord tissue from rats that received a contusion SCI. The N- and O-linked glycosylation was assessed at 3 and 14 days post-injury (DPI), and compared with uninjured control and time-matched sham spinal tissue. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS/MS) were performed to analyze carbohydrate structures. Results revealed diverse and abundant glycosylation in all groups, with some carbohydrate structures differentially produced in SCI animals compared with uninjured controls and shams. One such change occurred in the abundance of the Sda structure, Neu5Ac-α-(2,3)-[GalNAc-ß-(1,4)-]Gal-ß-(1,4)-GlcNAc, which was increased in SCI samples compared with shams and non-injured controls. Immunohistochemistry (IHC) and western blot were performed on SCI and sham samples using the CT1 antibody, which recognizes the terminal trisaccharide of Sda with high specificity. Both of these metrics confirmed elevated Sda structure in SCI tissue, where IHC further showed that Sda is expressed mainly by microglia. The results of these studies suggest that SCI causes a significant alteration in N- and O-linked glycosylation.

A 3-day exposure to 10% ethanol with 10% sucrose successfully initiates ethanol self-administration.

The initiation phase of ethanol self-administration is difficult to study using the well-established, sucrose-fading procedure due to the changing concentrations of ethanol in the first few days. The purpose of this experiment was to test whether a modified sucrose-substitution procedure in which rats are initially exposed to high concentrations of ethanol and sucrose for three days would successfully initiate ethanol self-administration. Male Long-Evans rats were trained to lever-press with a 10% sucrose solution in which four or 20 responses allowed 20-min access to the solution. Subsequently, rats were exposed to a 3-day period of operant self-administration of 10% sucrose+10% ethanol. This constant-concentration exposure was followed by the standard procedure in which sucrose is completely faded out. The establishment of ethanol self-administration was determined by ethanol intake, pre- and postprocedure two-bottle choice preference tests, and extinction trials. The mean ethanol intake was 2.2 times higher on day 2 compared with day 1 on the 10% sucrose+10% ethanol solution. After fading out the sucrose, the daily intake of 10% ethanol solution over 5 days was stable at approximately 0.57 g/kg. Ethanol preference was approximately threefold higher after the modified sucrose-fading procedure. Responding during a single session extinction test was dramatically increased from 4 to 61+/-13 or 20 to 112+/-22 responses in 20 min. Similar to the standard sucrose-fading method, we did not observe a significant relationship between extinction responding and ethanol intake. Blood alcohol concentrations were 4.5 mM 20 min after consumption began. We conclude that initiation and establishment of ethanol self-administration will occur using this modified sucrose-fading procedure.

Cerebellar Premotor Output Neurons Collateralize to Innervate the Cerebellar Cortex.

Motor commands computed by the cerebellum are hypothesized to use corollary discharge, or copies of outgoing commands, to accelerate motor corrections. Identifying sources of corollary discharge, therefore, is critical for testing this hypothesis. Here we verified that the pathway from the cerebellar nuclei to the cerebellar cortex in mice includes collaterals of cerebellar premotor output neurons, mapped this collateral pathway, and identified its postsynaptic targets. Following bidirectional tracer injections into a distal target of the cerebellar nuclei, the ventrolateral thalamus, we observed retrogradely labeled somata in the cerebellar nuclei and mossy fiber terminals in the cerebellar granule layer, consistent with collateral branching. Corroborating these observations, bidirectional tracer injections into the cerebellar cortex retrogradely labeled somata in the cerebellar nuclei and boutons in the ventrolateral thalamus. To test whether nuclear output neurons projecting to the red nucleus also collateralize to the cerebellar cortex, we used a Cre-dependent viral approach, avoiding potential confounds of direct red nucleus-to-cerebellum projections. Injections of a Cre-dependent GFP-expressing virus into Ntsr1-Cre mice, which express Cre selectively in the cerebellar nuclei, retrogradely labeled somata in the interposed nucleus, and putative collateral branches terminating as mossy fibers in the cerebellar cortex. Postsynaptic targets of all labeled mossy fiber terminals were identified using immunohistochemical Golgi cell markers and electron microscopic profiles of granule cells, indicating that the collaterals of nuclear output neurons contact both Golgi and granule cells. These results clarify the organization of a subset of nucleocortical projections that constitute an experimentally accessible corollary discharge pathway within the cerebellum.