Precerebellar Neurons Research Articles

Coexpression of VGLUT1 and VGLUT2 in precerebellar neurons in the lateral reticular nucleus of the rat

Vesicular glutamate transporter (VGLUT) 1 and VGLUT2 have been reported to distribute complementally in most brain regions and have been assumed to define distinct functional elements. Previous studies have shown the expression of VGLUT1 mRNA and VGLUT2 mRNA in the lateral reticular nucleus (LRN), a key precerebellar nucleus sending mossy fibers to the cerebellum. In the present study, we firstly examined the coexpression of VGLUT1 and VGLUT2 mRNA in the LRN of the rat by dual-fluorescence in situ hybridization. About 81.89 % of glutamatergic LRN neurons coexpressed VGLUT1 and VGLUT2 mRNA, and the others expressed either VGLUT1 or VGLUT2 mRNA. We then injected the retrograde tracer Fluogold (FG) into the vermal cortex of cerebellum, and observed that 95.01 % and 86.80 % of FG-labeled LRN neurons expressed VGLUT1 or VGLUT2 mRNA respectively. We further injected the anterograde tracer biotinylated dextran amine (BDA) into the LRN, and found about 82.6 % of BDA labeled axon terminals in the granular layer of cerebellar cortex showed both VGLUT1- and VGLUT2-immunoreactivities. Afterwards, we observed under electron microscopy that anterogradely labeled axon terminals showing immunoreactivity for VGLUT1 or VGLUT2 made asymmetric synapses with dendritic profiles of cerebellar neurons. Finally, we selectively down-regulated the expression of VGLUT1 mRNA or VGLUT2 mRNA by using viral vector mediated siRNA transfection and detected that the fine movements of the forelimb of rats were disturbed. These results indicated that LRN neurons coexpressing VGLUT1 and VGLUT2 project to the cerebellar cortex and these neurons might be critical in mediating the forelimb movements.

Postmitotic Hoxa5 Expression Specifies Pontine Neuron Positional Identity and Input Connectivity of Cortical Afferent Subsets

The mammalian precerebellar pontine nucleus (PN) has a main role in relaying cortical information to the cerebellum. The molecular determinants establishing ordered connectivity patterns between cortical afferents and precerebellar neurons are largely unknown. We show that expression of Hox5 transcription factors is induced in specific subsets of postmitotic PN neurons at migration onset. Hox5 induction is achieved by response to retinoic acid signaling, resulting in Jmjd3-dependent derepression of Polycomb chromatin and 3D conformational changes. Hoxa5 drives neurons to settle posteriorly in the PN, where they are monosynaptically targeted by cortical neuron subsets mainly carrying limb somatosensation. Furthermore, Hoxa5 postmigratory ectopic expression in PN neurons is sufficient to attract cortical somatosensory inputs regardless of position and avoid visual afferents. Transcriptome analysis further suggests that Hoxa5 is involved in circuit formation. Thus, Hoxa5 coordinates postmitotic specification, migration, settling position, and sub-circuit assembly of PN neuron subsets in the cortico-cerebellar pathway.

Tracing of Afferent Connections in the Zebrafish Cerebellum Using Recombinant Rabies Virus.

The cerebellum is involved in some forms of motor coordination and learning, and in cognitive and emotional functions. To elucidate the functions of the cerebellum, it is important to unravel the detailed connections of the cerebellar neurons. Although the cerebellar neural circuit structure is generally conserved among vertebrates, it is not clear whether the cerebellum receives and processes the same or similar information in different vertebrate species. Here, we performed monosynaptic retrograde tracing with recombinant rabies viruses (RV) to identify the afferent connections of the zebrafish cerebellar neurons. We used a G-deleted RV that expressed GFP. The virus was also pseudotyped with EnvA, an envelope protein of avian sarcoma and leucosis virus (ALSV-A). For the specific infection of cerebellar neurons, we expressed the RV glycoprotein (G) gene and the envelope protein TVA, which is the receptor for EnvA, in Purkinje cells (PCs) or granule cells (GCs), using the promoter for aldolase Ca (aldoca) or cerebellin 12 (cbln12), respectively. When the virus infected PCs in the aldoca line, GFP was detected in the PCs’ presynaptic neurons, including GCs and neurons in the inferior olivary nuclei (IOs), which send climbing fibers (CFs). These observations validated the RV tracing method in zebrafish. When the virus infected GCs in the cbln12 line, GFP was again detected in their presynaptic neurons, including neurons in the pretectal nuclei, the nucleus lateralis valvulae (NLV), the central gray (CG), the medial octavolateralis nucleus (MON), and the descending octaval nucleus (DON). GFP was not observed in these neurons when the virus infected PCs in the aldoca line. These precerebellar neurons generally agree with those reported for other teleost species and are at least partly conserved with those in mammals. Our results demonstrate that the RV system can be used for connectome analyses in zebrafish, and provide fundamental information about the cerebellar neural circuits, which will be valuable for elucidating the functions of cerebellar neural circuits in zebrafish.

The zinc-finger transcription factor GLI3 is a regulator of precerebellar neuronal migration.

Hindbrain precerebellar neurons arise from progenitor pools at the dorsal edge of the embryonic hindbrain: the caudal rhombic lip. These neurons follow distinct migratory routes to establish nuclei that provide climbing or mossy fiber inputs to the cerebellum. Gli3, a zinc-finger transcription factor in the Sonic hedgehog signaling pathway, is an important regulator of dorsal brain development. We demonstrate that in Gli3-null mutant mice, disrupted neuronal migratory streams lead to a disorganization of precerebellar nuclei. Precerebellar progenitors are properly established in Gli3-null embryos and, using conditional gene inactivation, we provide evidence that Gli3 does not play a cell-autonomous role in migrating precerebellar neurons. Thus, GLI3 likely regulates the development of other hindbrain structures, such as non-precerebellar nuclei or cranial ganglia and their respective projections, which may in turn influence precerebellar migration. Although the organization of non-precerebellar hindbrain nuclei appears to be largely unaffected in absence of Gli3, trigeminal ganglia and their central descending tracts are disrupted. We show that rostrally migrating precerebellar neurons are normally in close contact with these tracts, but are detached in Gli3-null embryos.

Non-cell autonomous control of precerebellar neuron migration by Slit and Robo proteins.

During development, precerebellar neurons migrate tangentially from the dorsal hindbrain to the floor plate. Their axons cross it but their cell bodies stop their ventral migration upon reaching the midline. It has previously been shown that Slit chemorepellents and their receptors, Robo1 and Robo2, might control the migration of precerebellar neurons in a repulsive manner. Here, we have used a conditional knockout strategy in mice to test this hypothesis. We show that the targeted inactivation of the expression of Robo1 and Robo2 receptors in precerebellar neurons does not perturb their migration and that they still stop at the midline. The selective ablation of the expression of all three Slit proteins in floor-plate cells has no effect on pontine neurons and only induces the migration of a small subset of inferior olivary neurons across the floor plate. Likewise, we show that the expression of Slit proteins in the facial nucleus is dispensable for pontine neuron migration. Together, these results show that Robo1 and Robo2 receptors act non-cell autonomously in migrating precerebellar neurons and that floor-plate signals, other than Slit proteins, must exist to prevent midline crossing.

Commissural neurons transgress the CNS/PNS boundary in absence of ventricular zone-derived netrin 1.

During the development of the central nervous system (CNS), only motor axons project into peripheral nerves. Little is known about the cellular and molecular mechanisms that control the development of a boundary at the CNS surface and prevent CNS neuron emigration from the neural tube. It has previously been shown that a subset of spinal cord commissural axons abnormally invades sensory nerves in Ntn1 hypomorphic embryos and Dcc knockouts. However, whether netrin 1 also plays a similar role in the brain is unknown. In the hindbrain, precerebellar neurons migrate tangentially under the pial surface, and their ventral migration is guided by netrin 1. Here, we show that pontine neurons and inferior olivary neurons, two types of precerebellar neurons, are not confined to the CNS in Ntn1 and Dcc mutant mice, but that they invade the trigeminal, auditory and vagus nerves. Using a Ntn1 conditional knockout, we show that netrin 1, which is released at the pial surface by ventricular zone progenitors is responsible for the CNS confinement of precerebellar neurons. We propose, that netrin 1 distribution sculpts the CNS boundary by keeping CNS neurons in netrin 1-rich domains.

Conditional Loss of Hoxa5 Function Early after Birth Impacts on Expression of Genes with Synaptic Function.

Hoxa5 is a member of the Hox gene family that plays critical roles in successive steps of the central nervous system formation during embryonic and fetal development. In the mouse, Hoxa5 was recently shown to be expressed in the medulla oblongata and the pons from fetal stages to adulthood. In these territories, Hoxa5 transcripts are enriched in many precerebellar neurons and several nuclei involved in autonomic functions, while the HOXA5 protein is detected mainly in glutamatergic and GABAergic neurons. However, whether HOXA5 is functionally required in these neurons after birth remains unknown. As a first approach to tackle this question, we aimed at determining the molecular programs downstream of the HOXA5 transcription factor in the context of the postnatal brainstem. A comparative transcriptomic analysis was performed in combination with gene expression localization, using a conditional postnatal Hoxa5 loss-of-function mouse model. After inactivation of Hoxa5 at postnatal days (P)1–P4, we established the transcriptome of the brainstem from P21 Hoxa5 conditional mutants using RNA-Seq analysis. One major finding was the downregulation of several genes associated with synaptic function in Hoxa5 mutant specimens including different actors involved in glutamatergic synapse, calcium signaling pathway, and GABAergic synapse. Data were confirmed and extended by reverse transcription quantitative polymerase chain reaction analysis, and the expression of several HOXA5 candidate targets was shown to co-localize with Hoxa5 transcripts in precerebellar nuclei. Together, these new results revealed that HOXA5, through the regulation of key actors of the glutamatergic/GABAergic synapses and calcium signaling, might be involved in synaptogenesis, synaptic transmission, and synaptic plasticity of the cortico-ponto-cerebellar circuitry in the postnatal brainstem.

HOXA5 localization in postnatal and adult mouse brain is suggestive of regulatory roles in postmitotic neurons.

Hoxa5 is a member of the Hox gene family, which plays critical roles in successive steps of the central nervous system formation during embryonic and fetal development. Hoxa5 expression in the adult mouse brain has been reported, suggesting that this gene may be functionally required in the brain after birth. To provide further insight into the Hoxa5 expression pattern and potential functions in the brain, we have characterized its neuroanatomical profile from embryonic stages to adulthood. While most Hox mapping studies have been based solely on transcript analysis, we extended our analysis to HOXA5 protein localization in adulthood using specific antibodies. Our results show that Hoxa5 expression appears in the most caudal part of the hindbrain at fetal stages, where it is maintained until adulthood. In the medulla oblongata and pons, we detected Hoxa5 expression in many precerebellar neurons and in several nuclei implicated in the control of autonomic functions. In these territories, the HOXA5 protein is present solely in neurons, specifically in γ-aminobutyric acid (GABA)ergic, glutamatergic, and catecholaminergic neurons. Finally, we also detected Hoxa5 transcripts, but not the HOXA5 protein, in the thalamus and the cortex, from postnatal stages to adult stages, and in the cerebellum at adulthood. We provide evidence that some larger variants of Hoxa5 transcripts are present in these territories. Our mapping analysis allowed us to build hypotheses regarding HOXA5 functions in the nervous system after birth, such as a potential role in the establishment and refinement/plasticity of precerebellar circuits during postnatal and adult life. J. Comp. Neurol. 525:1155-1175, 2017. © 2016 Wiley Periodicals, Inc.

Calm1 signaling pathway is essential for the migration of mouse precerebellar neurons

The calcium ion regulates many aspects of neuronal migration, which is an indispensable process in the development of the nervous system. Calmodulin (CaM) is a multifunctional calcium ion sensor that transduces much of the signal. To better understand the role of Ca(2+)-CaM in neuronal migration, we investigated mouse precerebellar neurons (PCNs), which undergo stereotyped, long-distance migration to reach their final position in the developing hindbrain. In mammals, CaM is encoded by three non-allelic CaM (Calm) genes (Calm1, Calm2 and Calm3), which produce an identical protein with no amino acid substitutions. We found that these CaM genes are expressed in migrating PCNs. When the expression of CaM from this multigene family was inhibited by RNAi-mediated acute knockdown, inhibition of Calm1 but not the other two genes caused defective PCN migration. Many PCNs treated with Calm1 shRNA failed to complete their circumferential tangential migration and thus failed to reach their prospective target position. Those that did reach the target position failed to invade the depth of the hindbrain through the required radial migration. Overall, our results suggest the participation of CaM in both the tangential and radial migration of PCNs.

The spino-bulbar-cerebellar pathway: organization and neurochemical properties of spinal cells that project to the lateral reticular nucleus in the rat.

In addition to classical spinocerebellar pathways, the cerebellum receives information from the spinal cord indirectly via spino-bulbar-cerebellar systems. One of the structures in this pathway is the lateral reticular nucleus (LRt). We performed series of experiments to investigate the organization and neurotransmitter content of spinoreticular tract (SRT) neurons in the lumbar spinal cord that project to the LRt. Three rats received injections of the b subunit of Cholera toxin (CTb) or Fluorogold (FG) within the left and right LRt. The majority of SRT cells (56–61%) were found within the contralateral medial intermediate gray matter where small numbers (7–10%) of double-labeled cells were also present on both sides of the cord. Six rats received unilateral spinal injections of CTb to label spinal projections to the LRt. Injections of FG were made also into the anterior lobe of the cerebellum to label LRt pre-cerebellar neurons. Terminals were found mainly ipsilateral to spinal injection sites within the central and ventrolateral regions of the LRt. Immunocytochemical analysis of SRT terminals revealed that the majority (75%) were contained vesicular glutamate transporter 2 but a minority (20%) contained the vesicular GABA transporter. The inhibitory subpopulation was found to be GABAergic, glycinergic, or contained both transmitters. Inhibitory and excitatory terminals were present within overlapping regions of the nucleus. Most CTb terminals contacting LRt pre-cerebellar neurons were excitatory (80%) whereas a minority were inhibitory and most cells (88%) received contacts from both inhibitory and excitatory terminals. This study shows that SRT axons in the LRt have the capacity to exert direct excitatory and inhibitory actions on LRt pre-cerebellar neurons. Thus spinal cord input has the capacity to facilitate or depress the activity of individual LRt cells which in turn adjust activity in the cerebellum to produce coordinated motor behaviors.

Calm1 signaling pathway is essential for the migration of mouse precerebellar neurons.

The calcium ion regulates many aspects of neuronal migration, which is an indispensable process in the development of the nervous system. Calmodulin (CaM) is a multifunctional calcium ion sensor that transduces much of the signal. To better understand the role of Ca(2+)-CaM in neuronal migration, we investigated mouse precerebellar neurons (PCNs), which undergo stereotyped, long-distance migration to reach their final position in the developing hindbrain. In mammals, CaM is encoded by three non-allelic CaM (Calm) genes (Calm1, Calm2 and Calm3), which produce an identical protein with no amino acid substitutions. We found that these CaM genes are expressed in migrating PCNs. When the expression of CaM from this multigene family was inhibited by RNAi-mediated acute knockdown, inhibition of Calm1 but not the other two genes caused defective PCN migration. Many PCNs treated with Calm1 shRNA failed to complete their circumferential tangential migration and thus failed to reach their prospective target position. Those that did reach the target position failed to invade the depth of the hindbrain through the required radial migration. Overall, our results suggest the participation of CaM in both the tangential and radial migration of PCNs.

Acute focal brain damage alters mitochondrial dynamics and autophagy in axotomized neurons.

Mitochondria are key organelles for the maintenance of life and death of the cell, and their morphology is controlled by continual and balanced fission and fusion dynamics. A balance between these events is mandatory for normal mitochondrial and neuronal function, and emerging evidence indicates that mitochondria undergo extensive fission at an early stage during programmed cell death in several neurodegenerative diseases. A pathway for selective degradation of damaged mitochondria by autophagy, known as mitophagy, has been described, and is of particular importance to sustain neuronal viability. In the present work, we analyzed the effect of autophagy stimulation on mitochondrial function and dynamics in a model of remote degeneration after focal cerebellar lesion. We provided evidence that lesion of a cerebellar hemisphere causes mitochondria depolarization in axotomized precerebellar neurons associated with PTEN-induced putative kinase 1 accumulation and Parkin translocation to mitochondria, block of mitochondrial fusion by Mfn1 degradation, increase of calcineurin activity and dynamin-related protein 1 translocation to mitochondria, and consequent mitochondrial fission. Here we suggest that the observed neuroprotective effect of rapamycin is the result of a dual role: (1) stimulation of autophagy leading to damaged mitochondria removal and (2) enhancement of mitochondria fission to allow their elimination by mitophagy. The involvement of mitochondrial dynamics and mitophagy in brain injury, especially in the context of remote degeneration after acute focal brain damage, has not yet been investigated, and these findings may offer new target for therapeutic intervention to improve functional outcomes following acute brain damage.

Spinal cord projections to the cerebellum in the mouse.

The projections from the spinal cord to the cerebellar cortex were studied using retrograde neuronal tracers. Thus far, no study has shown the detailed topographic mapping of the projections from the spinal neuron clusters to the cerebellar cortex regions for experimental animals, and there are no studies for the mouse. Tracers Fluoro-Gold and cholera toxin B were injected into circumscribed regions of the cerebellar cortex, and retrogradely labeled spinal cord neurons were mapped throughout the spinal cord. Spinal projections to the cerebellar cortex were mainly from five neuronal columns--central cervical nucleus, dorsal nucleus, lumbar and sacral precerebellar nuclei, and lumbar border precerebellar cells--and from scattered neurons located in the deep dorsal horn and laminae 6-8. The spinocerebellar projections to the cortex were mainly to the vermis. All five precerebellar cell columns projected to both anterior and posterior parts of the cerebellar cortex. Results of this study provide an amendment to the known rostral and caudal boundaries of the precerebellar cell columns in the mouse. Scattered precerebellar neurons in the most caudal deep dorsal horn and laminae 6-8 projected exclusively to the anterior part of the cerebellar cortex. In this study, no labeled spinal neurons were found to project to the lobules 6 and 7 of the cerebellar vermis, the flocculus, and the paraflocculus. Spinocerebellar neurons were located bilaterally, but the majority of the projections were contralateral for the central cervical nucleus, and ipsilateral for the remaining spinal precerebellar neuronal clusters.

Inhibitory effects of draxin on axonal outgrowth and migration of precerebellar neurons

The rhombic lip, a dorsal stripe of the neuroepithelium lining the edge of the fourth ventricle, is the site of origin of precerebellar neurons (PCN), which migrate tangentially towards the floor plate. After reaching the floor plate, they project their axons to the cerebellum. Although previous studies have shown that the guidance molecules Netrin/DCC and Slit/Robo have critical roles in PCN migration, the molecular mechanisms underlying this process remain poorly understood. Here, we report that draxin, a repulsive axon guidance protein, is involved in PCN development. We found that draxin is expressed in the rhombic lip and migratory stream of some PCN in the developing hindbrain of mice. In addition, draxin inhibited neurite outgrowth and nuclei migration from rhombic lip explants. These results suggest that draxin functions as a repulsive guidance cue for PCN migration. However, we observed no significant differences in PCN distribution between draxin−/− and wild type embryos. Thus, draxin and other axon guidance cues may have redundant roles in PCN migration.

Skilled reaching relies on a V2a propriospinal internal copy circuit

The precision of skilled forelimb movement has long been presumed to rely on rapid feedback corrections triggered by internally-directed copies of outgoing motor commands – but the functional relevance of inferred internal copy circuits has remained unclear. One class of spinal interneurons implicated in the control of mammalian forelimb movement, cervical propriospinal neurons (PNs), has the potential to convey an internal copy of pre-motor signals through dual innervation of forelimb-innervating motor neurons and pre-cerebellar neurons of the lateral reticular nucleus. We have examined whether the PN internal copy pathway functions in the control of goal-directed reaching. In mice, PNs include a genetically-accessible subpopulation of cervical V2a interneurons, and their targeted ablation perturbs reaching while leaving intact other elements of forelimb movement. Moreover, optogenetic activation of the PN internal copy branch recruits a rapid cerebellar feedback loop that modulates forelimb motor neuron activity and severely disrupts reaching kinematics. Our findings implicate V2a PNs as the focus of an internal copy pathway assigned to the rapid updating of motor output during reaching behavior.

The control of precerebellar neuron migration by RNA-binding protein Csde1

Neuronal migration during brain development sets the position of neurons for the subsequent wiring of neural circuits. To understand the molecular mechanism regulating the migrating process, we considered the migration of mouse precerebellar neurons. Precerebellar neurons originate in the rhombic lip of the hindbrain and show stereotypic, long-distance tangential migration along the circumference of the hindbrain to form precerebellar nuclei at discrete locations. To identify the molecular components underlying this navigation, we screened for genes expressed in the migrating precerebellar neurons. As a result, we identified the following three genes through the screening; Calm1, Septin 11, and Csde1. We report here functional analysis of one of these genes, Csde1, an RNA-binding protein implicated in the post-transcriptional regulation of a subset of cellular mRNA, by examining its participation in precerebellar neuronal migration. We found that shRNA-mediated inhibition of Csde1 expression resulted in a failure of precerebellar neurons to complete their migration into their prospective target regions, with many neurons remaining in migratory paths. Furthermore, those that did reach their destination failed to invade the depth of the hindbrain via radial migration. These results have uncovered a crucial role of Csde1 in the proper control of both radial and tangential migration of precerebellar neurons.

Evidence That Adrenergic Ventrolateral Medullary Cells Are Activated whereas Precerebellar Lateral Reticular Nucleus Neurons Are Suppressed during REM Sleep

Rapid eye movement sleep (REMS) is generated in the brainstem by a distributed network of neurochemically distinct neurons. In the pons, the main subtypes are cholinergic and glutamatergic REMS-on cells and aminergic REMS-off cells. Pontine REMS-on cells send axons to the ventrolateral medulla (VLM), but little is known about REMS-related activity of VLM cells. In urethane-anesthetized rats, dorsomedial pontine injections of carbachol trigger REMS-like episodes that include cortical and hippocampal activation and suppression of motoneuronal activity; the episodes last 4–8 min and can be elicited repeatedly. We used this model to determine whether VLM catecholaminergic cells are silenced during REMS, as is typical of most aminergic neurons studied to date, and to investigate other REMS-related cells in this region. In 18 anesthetized, paralyzed and artificially ventilated rats, we obtained extracellular recordings from VLM cells when REMS-like episodes were elicited by pontine carbachol injections (10 mM, 10 nl). One major group were the cells that were activated during the episodes (n = 10). Their baseline firing rate of 3.7±2.1 (SD) Hz increased to 9.7±2.1 Hz. Most were found in the adrenergic C1 region and at sites located less than 50 µm from dopamine β-hydroxylase-positive (DBH+) neurons. Another major group were the silenced or suppressed cells (n = 35). Most were localized in the lateral reticular nucleus (LRN) and distantly from any DBH+ cells. Their baseline firing rates were 6.8±4.4 Hz and 15.8±7.1 Hz, respectively, with the activity of the latter reduced to 7.4±3.8 Hz. We conclude that, in contrast to the pontine noradrenergic cells that are silenced during REMS, medullary adrenergic C1 neurons, many of which drive the sympathetic output, are activated. Our data also show that afferent input transmitted to the cerebellum through the LRN is attenuated during REMS. This may distort the spatial representation of body position during REMS.

The spinal precerebellar nuclei: Calcium binding proteins and gene expression profile in the mouse

We have localized the spinocerebellar neuron groups in C57BL/6J mice by injecting the retrograde neuronal tracer Fluoro-Gold into the cerebellum and examined the distribution of SMI 32 and the calcium-binding proteins (CBPs), calbindin-D-28K (Cb), calretinin (Cr), and parvalbumin (Pv) in the spinal precerebellar nuclei. The spinal precerebellar neuron clusters identified were the dorsal nucleus, central cervical nucleus, lumbar border precerebellar nucleus, lumbar precerebellar nucleus, and sacral precerebellar nucleus. Some dispersed neurons in the deep dorsal horn and spinal laminae 6–8 also projected to the cerebellum. Cb, Cr, Pv, and SMI 32 were present in all major spinal precerebellar nuclei and Pv was the most commonly observed CBP. A number of genes expressed in hindbrain precerebellar nuclei are also expressed in spinal precerebellar groups, but there were some differences in gene expression profile between the different spinal precerebellar nuclei, pointing to functional diversity amongst them.

Stimulation of autophagy by rapamycin protects neurons from remote degeneration after acute focal brain damage

Autophagy is the evolutionarily conserved degradation and recycling of cellular constituents. In mammals, autophagy is implicated in the pathogenesis of many neurodegenerative diseases. However, its involvement in acute brain damage is unknown. This study addresses the function of autophagy in neurodegeneration that has been induced by acute focal cerebellar lesions. We provide morphological, ultrastructural, and biochemical evidence that lesions in a cerebellar hemisphere activate autophagy in axotomized precerebellar neurons. Through time course analyses of the apoptotic cascade, we determined mitochondrial dysfunction to be the early trigger of degeneration. Further, the stimulation of autophagy by rapamycin and the employment of mice with impaired autophagic responses allowed us to demonstrate that autophagy protects from damage promoting functional recovery. These findings have therapeutic significance, demonstrating the potential of pro-autophagy treatments for acute brain pathologies, such as stroke and brain trauma.

Distinct regulation of nNOS and iNOS by CB2 receptor in remote delayed neurodegeneration

Hemicerebellectomy results in remote delayed degeneration of precerebellar neurons. We have reported that such a lesion induces type 2 cannabinoid receptor (CB(2)) expression in precerebellar neurons and that stimulation of CB(2), but not CB(1), has neuroprotective effects. In this study, we found that in the same model, the CB(2) agonist JWH-015 enhances neuronal nitric oxide synthase (nNOS) expression in axotomized neurons and that CB(2)-mediated neuroprotection is abrogated by pharmacological inhibition of nNOS. JWH-015 prevented the axotomy-induced upregulation of inducible NOS (iNOS) in astrocytes but had no effect on endothelial NOS (eNOS). In addition, we observed that JWH-015 significantly reduces hemicerebellectomy-induced neuroinflammatory responses and oxidative/nitrative stress. With regard to the signaling pathways of CB(2)/nNOS-mediated neuroprotection, we noted nNOS-dependent modulation of the expression of anti-oxidative (Hsp70) and anti-apoptotic (Bcl-2) proteins. These findings shed light on the interactions between the endocannabinoid and nitrergic systems after focal brain injury, implicating distinct functions of nNOS activation and iNOS inhibition in CB(2) signaling, which protect neurons from axotomy-induced cell death.