Fasciculus Retroflexus Research Articles

The Neuroanatomy of the Habenular Complex and Its Role in the Regulation of Affective Behaviors.

The habenular complex is a diencephalic structure divided into the medial and lateral divisions that lie within the epithalamus of most vertebrates. This brain structure, whose activities are mainly regulated via inputs/outputs from and to the stria medullaris and the fasciculus retroflexus, plays a significant role in the modulation of anti-reward behaviors in both the rodent and human brain. Such anti-reward circuits are regulated by dopaminergic and serotonergic projections with several other subcortical and cortical regions; therefore, it is plausible that impairment to this key subcortical structure or its connections contributes to the pathogenesis of affective disorders. Current literature reveals the existence of structural changes in the habenula complex in individuals afflicted by such disorders; however, there is a need for more comprehensive investigations to elucidate the underlying neuroanatomical connections that underpin disease development. In this review article, we aim to provide a comprehensive view of the neuroanatomical differences between the rodent and human habenular complex, the main circuitries, and provide an update on the emerging roles of this understudied subcortical structure in the control of affective behaviors, with special emphasis to morbid conditions of the affective sphere.

Characterization of Vps13b-mutant mice reveals neuroanatomical and behavioral phenotypes with females less affected

The vacuolar protein sorting-associated protein 13B (VPS13B) is a large and highly conserved protein. Disruption of VPS13B causes the autosomal recessive Cohen syndrome, a rare disorder characterized by microcephaly and intellectual disability among other features, including developmental delay, hypotonia, and friendly-personality. However, the underlying mechanisms by which VPS13B disruption leads to brain dysfunction still remain unexplained.To gain insights into the neuropathogenesis of Cohen syndrome, we systematically characterized brain changes in Vps13b-mutant mice and compared murine findings to 235 previously published and 17 new patients diagnosed with VPS13B-related Cohen syndrome.We showed that Vps13b is differentially expressed across brain regions with the highest expression in the cerebellum, the hippocampus and the cortex with postnatal peak. Half of the Vps13b−/− mice die during the first week of life. The remaining mice have a normal lifespan and display the core phenotypes of the human disease, including microcephaly, growth delay, hypotonia, altered memory, and enhanced sociability. Systematic 2D and 3D brain histo-morphological analyses reveal specific structural changes in the brain starting after birth. The dentate gyrus is the brain region with the most prominent reduction in size, while the motor cortex is specifically thinner in layer VI. The fornix, the fasciculus retroflexus, and the cingulate cortex remain unaffected. Interestingly, these neuroanatomical changes implicate an increase of neuronal death during infantile stages with no progression in adulthood suggesting that VPS13B promotes neuronal survival early in life. Importantly, whilst both sexes were affected, some neuroanatomical and behavioral phenotypes were less pronounced or even absent in females. We evaluate sex differences in Cohen patients and conclude that females are less affected both in mice and patients.Our findings provide new insights about the neurobiology of VPS13B and highlight previously unreported brain phenotypes while defining Cohen syndrome as a likely new entity of non-progressive infantile neurodegeneration.

Nuclei and tracts in the epithalamus of crocodiles.

The epithalamus, an area of the dorsal diencephalon found in all vertebrates, consists of the habenula, the subhabenular nuclei, and associated tracts. The habenula is itself divisible into two parts-a medial and a lateral nucleus differing in theirinputs, outputs, and cellular morphology. The medial component is related to the limbic system and serotonergic raphe, while the lateral nucleus is more interconnected with the basal ganglia and midbrain dopamine systems. These findings, which come from experiments mainly done on mammals, serve as a basis for comparison with other vertebrates. However, similar studies in other amniotes, such as reptiles, are few. To fill this gap in knowledge, two species of crocodiles were examined utilizing a variety of histological methods in various planes of section. The following results were obtained. First, the habenula was divided into medial and lateral parts based on its cytoarchitecture. Neurons in the medial habenula were small, were closely packed, and had a limited dendritic arbor characterized by unusual distal dendritic appendages, whereas neurons in the lateral habenula were larger, were more loosely packed, and had longer dendritic processes that were commonly beaded. Second, the stria medullaris, the major input to the habenula, was identified by its immunoreactivity to parvalbumin. Third, the fasciculus retroflexus (habenulointerpeduncular tract), the primary output of the habenula, was visualized by staining with acetylcholinesterase. Fourth, nuclei associated with the habenula, the subhabenular nuclei, have been identified and characterized. These features provide a means to recognize the major nuclei and tracts in the epithalamus in crocodiles and are likely applicable to other reptiles.

A disynaptic basal ganglia connection to the inferior olive: potential for basal ganglia influence on cerebellar learning.

Recent studies have shown that the cerebellum and the basal ganglia are interconnected at subcortical levels. However, a subcortical basal ganglia connection to the inferior olive (IO), being the source of the olivocerebellar climbing fiber system, is not known. We have used classical tracing with CTb, retrograde transneuronal infection with wildtype rabies virus, conditional tracing with genetically modified rabies virus, and examination of material made available by the Allen Brain Institute, to study potential basal ganglia connections to the inferior olive in rats and mice. We show in both species that parvalbumin-positive, and therefore GABAergic, neurons in the entopeduncular nucleus, representing the rodent equivalent of the internal part of the globus pallidus, innervate a group of cells that surrounds the fasciculus retroflexus and that are collectively known as the area parafascicularis prerubralis. As these neurons supply a direct excitatory input to large parts of the inferior olivary complex, we propose that the entopeduncular nucleus, as a main output station of the basal ganglia, provides an inhibitory influence on olivary excitability. As such, this connection may influence olivary involvement in cerebellar learning and/or could be involved in transmission of reward properties that have recently been established for olivocerebellar signaling.

Pharmacological evaluation of lateral habenula and rostromedial tegmental nucleus in the expression of ethanol-induced place preference.

Although ethanol administration produces a range of physiological effects, the rewarding aspect associated with its consumption is a major contributory factor to its abuse liability. Recently, lateral habenula (LHb) has been shown to be engaged by both rewarding and aversive stimuli. Its major glutamatergic output, the fasciculus retroflexus, projects to the rostromedial tegmental nucleus (RMTg) and controls the activity of the ventral tegmental area (VTA) dopaminergic system to promote reward circuitry. While several attempts have been made to understand the relationship between LHb and addiction, there is still a lack of knowledge in relation to ethanol addiction. In the present study, by pharmacologically exacerbating or inhibiting the LHb or RMTg neuronal activity during a post-conditioning test, we investigated the role of LHb-RMTg fasciculus retroflexus in ethanol-induced reward behavior using the conditioned place preference (CPP) test. We found that activation of LHb glutamatergic system by intra-LHb administration of l-trans-2,4-pyrrolidine dicarboxylate (PDC) (glutamate transporter inhibitor) significantly decreased CPP score; on the contrary, lamotrigine (inhibits glutamate release) significantly increased CPP score and showed a rewarding effect in CPP. Instead, intra-RMTg administration of muscimol (GABAA receptor agonist) significantly increased CPP score, whereas bicuculline (GABAA antagonist) treatment decreased CPP score. In immunohistochemistry, we found that PDC administration significantly decreased, whereas lamotrigine treatment significantly increased tyrosine hydroxylase immunoreactivity (TH-ir) in VTA and nucleus accumbens (NAc). Furthermore, while intra-RMTg administration of muscimol increased, the bicuculline treatment significantly decreased the TH-ir in VTA and NAc. Together, our behavioral and immunohistochemical results signify the role of LHb and RMTg in the expression of ethanol-conditioned reward behavior.

Adhesion molecule Amigo2 is involved in the fasciculation process of the fasciculus retroflexus.

The fasciculus retroflexus is the prominent efferent pathway from the habenular complex. Medial habenular axons form a core packet whereas lateral habenular axons course in a surrounding shell. Both groups of fibers share the same initial pathway but differ in the final segment of the tract, supposedly regulated by surface molecules. The gene Amigo2 codes for a membrane adhesion molecule with an immunoglobulin-like domain 2 and is selectively expressed in the medial habenula. We present it as a candidate for controlling the fasciculation behavior of medial habenula axons. First, we studied the development of the habenular efferents in an Amigo2 lack of function mouse model. The fasciculus retroflexus showed a variable defasciculation phenotype. Gain of function experiments allowed us to generate a more condensed tract and rescued the Amigo2 knock-out phenotype. Changes in Amigo2 function did not alter the course of habenular fibers. We have demonstrated that Amigo2 plays a subtle role in the fasciculation of the fasciculus retroflexus.

The lateral habenula is not required for ethanol dependence-induced escalation of drinking.

The lateral habenula (LHb) is an epithalamic nuclei that has been shown to signal the aversive properties of ethanol. The present study tested the hypothesis that activity of the LHb is required for the acquisition and/or expression of dependence-induced escalation of ethanol drinking and somatic withdrawal symptoms. Male Sprague-Dawley rats completed 4 weeks of baseline drinking under a standard intermittent access two-bottle choice (2BC) paradigm before undergoing 2 weeks of daily chronic intermittent ethanol (CIE) via vapor inhalation. Following this CIE exposure period, rats resumed 2BC drinking to assess dependence-induced changes in voluntary ethanol consumption. CIE exposed rats exhibited a significant increase in ethanol drinking that was associated with high levels of blood alcohol and a reduction in somatic symptoms of ethanol withdrawal. However, despite robust cFos activation in the LHb during ethanol withdrawal, chemogenetic inhibition of the LHb did not alter either ethanol consumption or somatic signs of ethanol withdrawal. Consistent with this observation, ablating LHb outputs via electrolytic lesions of the fasciculus retroflexus (FR) did not alter the acquisition of somatic withdrawal symptoms or escalation of ethanol drinking in CIE-exposed rats. The LHb controls activity of the rostromedial tegmental nucleus (RMTg), a midbrain nucleus activated by aversive experiences including ethanol withdrawal. During ethanol withdrawal, both FR lesioned and sham control rats exhibited similar cFos activation in the RMTg, suggesting that RMTg activation during ethanol withdrawal does not require LHb input. These data suggest that, at least in male rats, the LHb is not necessary for the acquisition or expression of escalation of ethanol consumption or expression of somatic symptoms of ethanol withdrawal. Overall, our findings provide evidence that the LHb is dispensable for some of the negative consequences of ethanol withdrawal.

Laterality and Sex Differences of Human Lateral Habenula Afferent and Efferent Fiber Tracts.

IntroductionThe lateral habenula (LHb) is an epithalamic nucleus associated with negative valence and affective disorders. It receives input via the stria medullaris (SM) and sends output via the fasciculus retroflexus (FR). Here, we use tractography to reconstruct and characterize this pathway.MethodsMulti-shell human diffusion magnetic resonance imaging (dMRI) data was obtained from the human connectome project (HCP) (n = 20, 10 males) and from healthy controls (n = 10, 6 males) scanned at our institution. We generated LHb afferents and efferents using probabilistic tractography by selecting the pallidum as the seed region and the ventral tegmental area as the output target.ResultsWe were able to reconstruct the intended streamlines in all individuals from the HCP dataset and our dataset. Our technique also aided in identification of the LHb. In right-handed individuals, the streamlines were significantly more numerous in the left hemisphere (mean ratio 1.59 ± 0.09, p = 0.04). In left-handed individuals, there was no hemispheric asymmetry on average (mean ratio 1.00 ± 0.09, p = 1.0). Additionally, these streamlines were significantly more numerous in females than in males (619.9 ± 159.7 vs. 225.9 ± 66.03, p = 0.04).ConclusionWe developed a method to reconstruct the SM and FR without manual identification of the LHb. This technique enables targeting of these fiber tracts as well as the LHb. Furthermore, we have demonstrated that there are sex and hemispheric differences in streamline number. These findings may have therapeutic implications and warrant further investigation.

Stria medullaris innervation follows the transcriptomic division of the habenula

The habenula is a complex neuronal population integrated in a pivotal functional position into the vertebrate limbic system. Its main afference is the stria medullaris and its main efference the fasciculus retroflexus. This neuronal complex is composed by two main components, the medial and lateral habenula. Transcriptomic and single cell RNAseq studies have unveiled the morphological complexity of both components. The aim of our work was to analyze the relation between the origin of the axonal fibers and their final distribution in the habenula. We analyzed 754 tracing experiments from Mouse Brain Connectivity Atlas, Allen Brain Map databases, and selected 12 neuronal populations projecting into the habenular territory. Our analysis demonstrated that the projections into the medial habenula discriminate between the different subnuclei and are generally originated in the septal territory. The innervation of the lateral habenula displayed instead a less restricted distribution from preoptic, terminal hypothalamic and peduncular nuclei. Only the lateral oval subnucleus of the lateral habenula presented a specific innervation from the dorsal entopeduncular nucleus. Our results unveiled the necessity of novel sorts of behavioral experiments to dissect the different functions associated with the habenular complex and their correlation with the distinct neuronal populations that generate them.

Neuromeric Distribution of Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase Activity in the Adult Lamprey Brain.

This study reports for the first time the distribution and morphological characterization of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d; a reliable marker of nitric oxide synthase activity) positive elements in the central nervous system of the adult river lamprey (Lampetra fluviatilis) on the framework of the neuromeric model and compares their cytoarchitectonic organization with that of gnathostomes. Both NADPH-d exhibiting cells and fibers were observed in all major divisions of the lamprey brain as well as in the spinal cord. In the secondary prosencephalon, NADPH-d positive cells were observed in the mitral cell layer of the olfactory bulb, evaginated pallium, amygdala, dorsal striatum, septum, lateral preoptic nucleus, caudal paraventricular area, posterior entopeduncular nucleus, nucleus of the stria medullaris, hypothalamic periventricular organ and mamillary region sensu lato. In the lamprey diencephalon, NADPH-d labeled cells were observed in several nuclei of the prethalamus, epithalamus, pretectum, and the basal plate. Especially remarkable was the staining observed in the right habenula and several pretectal nuclei. NADPH-d positive cells were also observed in the following mesencephalic areas: optic tectum (two populations), torus semicircularis, nucleus M5 of Schöber, and a ventral tegmental periventricular nucleus. Five different cell populations were observed in the isthmic region, whereas the large sensory dorsal cells, some cells located in the interpeduncular nucleus, the motor nuclei of most cranial nerves, the solitary tract nucleus, some cells of the reticular nuclei, and small cerebrospinal fluid-contacting (CSF-c) cells were the most evident stained cells of the rhombencephalon proper. Finally, several NADPH-d positive cells were observed in the rostral part of the spinal cord, including the large sensory dorsal cells, numerous CSF-c cells, and some dorsal and lateral interneurons. NADPH-d positive fibers were observed in the olfactory pathways (primary olfactory fibers and stria medullaris), the fasciculus retroflexus, and the dorsal column tract. Our results on the distribution of NADPH-d positive elements in the brain of the adult lamprey L. fluviatilis are significantly different from those previously reported in larval lampreys and demonstrated that these animals possess a complex nitrergic system readily comparable to those of other vertebrates, although important specific differences also exist.

Wnt1 Role in the Development of the Habenula and the Fasciculus Retroflexus.

Wnt1 is one of the morphogenes that controls the specification and differentiation of neuronal populations in the developing central nervous system. The habenula is a diencephalic neuronal complex located in the most dorsal aspect of the thalamic prosomere. This diencephalic neuronal population is involved in the limbic system and its malfunction is related with several psychiatric disorders. Our aim is to elucidate the Wnt1 role in the habenula and its main efferent tract, the fasciculus retroflexus, development. In order to achieve these objectives, we analyzed these structures development in a Wnt1 lack of function mouse model. The habenula was generated in our model, but it presented an enlarged volume. This alteration was due to an increment in habenular neuroblasts proliferation rate. The fasciculus retroflexus also presented a wider and disorganized distribution and a disturbed final trajectory toward its target. The mid-hindbrain territories that the tract must cross were miss-differentiated in our model. The specification of the habenula is Wnt1 independent. Nevertheless, it controls its precursors proliferation rate. Wnt1 expressed in the isthmic organizer is vital to induce the midbrain and rostral hindbrain territories. The alteration of these areas is responsible for the fasciculus retroflexus axons misroute.

Netrin 1-Mediated Role of the Substantia Nigra Pars Compacta and Ventral Tegmental Area in the Guidance of the Medial Habenular Axons.

The fasciculus retroflexus is an important fascicle that mediates reward-related behaviors and is associated with different psychiatric diseases. It is the main habenular efference and constitutes a link between forebrain regions, the midbrain, and the rostral hindbrain. The proper functional organization of habenular circuitry requires complex molecular programs to control the wiring of the habenula during development. However, the mechanisms guiding the habenular axons toward their targets remain mostly unknown. Here, we demonstrate the role of the mesodiencephalic dopaminergic neurons (substantia nigra pars compacta and ventral tegmental area) as an intermediate target for the correct medial habenular axons navigation along the anteroposterior axis. These neuronal populations are distributed along the anteroposterior trajectory of these axons in the mesodiencephalic basal plate. Using in vitro and in vivo experiments, we determined that this navigation is the result of netrin 1 attraction generated by the mesodiencephalic dopaminergic neurons. This attraction is mediated by the receptor deleted in colorectal cancer (DCC), which is strongly expressed in the medial habenular axons. The increment in our knowledge on the fasciculus retroflexus trajectory guidance mechanisms opens the possibility of analyzing if its alteration in mental health patients could account for some of their symptoms.

Diffusion Tensor Imaging: A Promising New Technique for Accurate Identification of the Stria Medullaris and Habenula

The Stria Medullaris (SM) is a white-matter tract that contains afferent fibres that connect the cognitive-emotional areas in the forebrain to the Habenula (Hb). The Hb plays an important role in behavioral responses to reward, stress, anxiety, pain, and sleep through its action on neuromodulator systems. The Fasciculus Retroflexus (FR) forms the primary output of the Hb to the midbrain. The SM, Hb, and FR are part of a special pathway between the forebrain and the midbrain known as the Dorsal Diencephalic Conduction system (DDC). Hb dysfunction is accompanied by different types of neuropsychiatric disorders, such as schizophrenia, depression, and Treatment-Resistant Depression (TRD). Due to difficulties in the imaging assessment of the SM and HB in vivo, they had not been a focus of clinical studies until the invention of Diffusion Tensor Imaging (DTI), which has revolutionized the imaging and investigation of the SM and Hb. DTI has facilitated the imaging of the SM and Hb and has provided insights into their properties through the investigation of their monoamine dysregulation. DTI is a well-established technique for mapping brain microstructure and white matter tracts; it provides indirect information about the microstructural architecture and integrity of white matter in vivo, based on water diffusion properties in the intra- and extracellular space, such as Axial Diffusivity (AD), Radial Diffusivity (RD), mean diffusivity, and Fractional Anisotropy (FA). Neurosurgeons have recognized the potential value of DTI in the direct anatomical targeting of the SM and Hb prior to Deep Brain Stimulation (DBS) surgery for the treatment of certain neuropsychiatric conditions, such as TRD. DTI is the only non-invasive method that offers the possibility of visualization in vivo of the white-matter tracts and nuclei in the human brain. This review study summarizes the use of DTI as a promising new imaging method for accurate identification of the SM and Hb, with special emphasis on direct anatomical targeting of the SM and Hb prior to DBS surgery.

Acupuncture reduces cocaine psychomotor responses by activating the rostromedial tegmental nucleus.

The rostromedial tegmental nucleus (RMTg), a GABAergic afferent to midbrain dopamine (DA) neurons, has emerged as an integral player in both rewarding and nociceptive responses. While previous studies have demonstrated that acupuncture modulates DA transmission in the mesolimbic reward system originating in the ventral tegmental area (VTA) and projecting to the nucleus accumbens (NAc) and can reduce drug self-administration, the central links between peripheral acupuncture signals and brain reward systems are not well-characterized. Thus, we hypothesised that acupuncture would elicit inhibitory signals from RMTg neurons to brain reward systems. Acupuncture reduced acute cocaine-induced locomotor activity and DA release in a point-specific manner, which was blocked by optogenetic silencing or chemical lesion of the RMTg. The acupuncture effect was mimicked by chemical activation of the RMTg. Acupuncture activated RMTg GABA neurons. In addition, the inhibitory effects of acupuncture on acute cocaine-induced locomotor activity were prevented by electrolytic lesions of the lateral habenula (LHb) or fasciculus retroflexus (FR), areas known to project to the RMTg. These findings suggest that acupuncture recruits the RMTg to reduce the psychomotor responses enhanced by acute cocaine.

A basic MRI anatomy of the rat brain in coronal sections for practical guidance to neuroscientists

Identification of the brain structures in the magnetic resonance imaging (MRI) of the rat is very important for the experimental work of many neuroscientists. Our intention was to recognize most of the structures without overlapping the MRI sections with the histological templates. Three live rats were used for this study who were examined in a micro-MRI apparatus by performing T2-weighted sequences in serial brain sections. Most of the white matter structures were easily identified, e.g. the anterior commissure, corpus callosum with forceps minor and major, cingulum, external and internal capsules, fornix, stria medullaris and terminalis, cranial nerves, mammillothalamic tract, fasciculus retroflexus, medial and lateral lemniscus, posterior commissure, commissures of the superior and inferior colliculi, medial longitudinal fasciculus, and the cerebral peduncle. Large and small gray matter structures were recognized as well, for example, the anterior olfactory structures, nucleus accumbens, caudate putamen, claustrum, bed nucleus of the stria terminalis, pituitary gland, globus pallidus, amygdala, some midline and intralaminar thalamic nuclei, certain hypothalamic nuclei, hippocampal formation, pineal body, periaqueductal gray matter, lateral and medial geniculate bodies, superior and inferior colliculi, and cranial nerves nuclei. All in all, of the total 160 recognized brain structures, 77 were identified without using the corresponding histological atlases. We believe that our labeled MRI pictures could be an important way for quick orientation for evaluating the effects of the experimental work regarding the rat brain.

Brain Iron Accumulation and the Formation of Calcifications After Developmental Zika Virus Infection.

Intracranial calcifications (ICC) are the most common neuropathological finding in the brains of children exposed in utero to the Zika virus (ZIKV). Using a mouse model of developmental ZIKV infection, we reported widespread calcifications in the brains of susceptible mice that correlated in multiple ways with the behavioral deficits observed. Here, we examined the time course of ICC development and the role of iron deposition in this process, in 3 genetically distinct inbred strains of mice. Brain iron deposits were evident by Perls' staining at 2 weeks post infection, becoming increasingly dense and coinciding with calcium buildup and the formation of ICCs. A regional analysis of the brains of susceptible mice (C57BL/6J and 129S1/SvImJ strains) revealed the presence of iron initially in regions containing many ZIKV-immunoreactive cells, but then spreading to regions containing few infected cells, most notably the thalamus and the fasciculus retroflexus. Microglial activation was widespread initially and later delineated the sites of ICC formation. Behavioral tests conducted at 5-6 weeks of age revealed greater deficits in mice with the most extensive iron deposition and calcification of subcortical regions, such as thalamus. These findings point to iron deposition as a key factor in the development of ICCs after developmental ZIKV infection.

Untangling the dorsal diencephalic conduction system: a review of structure and function of the stria medullaris, habenula and fasciculus retroflexus.

The often-overlooked dorsal diencephalic conduction system (DDCS) is a highly conserved pathway linking the basal forebrain and the monoaminergic brainstem. It consists of three key structures; the stria medullaris, the habenula and the fasciculus retroflexus. The first component of the DDCS, the stria medullaris, is a discrete bilateral tract composed of fibers from the basal forebrain that terminate in the triangular eminence of the stalk of the pineal gland, known as the habenula. The habenula acts as a relay hub where incoming signals from the stria medullaris are processed and subsequently relayed to the midbrain and hindbrain monoaminergic nuclei through the fasciculus retroflexus. As a result of its wide-ranging connections, the DDCS has recently been implicated in a wide range of behaviors related to reward processing, aversion and motivation. As such, an understanding of the structure and connections of the DDCS may help illuminate the pathophysiology of neuropsychiatric disorders such as depression, addiction and pain. This is the first review of all three components of the DDCS, the stria medullaris, the habenula and the fasciculus retroflexus, with particular focus on their anatomy, function and development.

Abstract #114: Rapidly reversible behavioral arrest during fasciculus retroflexus deep brain stimulation in a healthy non-human primate

Abstract #114: Rapidly reversible behavioral arrest during fasciculus retroflexus deep brain stimulation in a healthy non-human primate

Isotropic 25-Micron 3D Neuroimaging Using ex vivo Microstructural Manganese-Enhanced MRI (MEMRI).

MRI observations following in vivo administration of Mn2+ [manganese (Mn)-enhanced MRI, MEMRI] have been used as an excellent morphological and functional MRI tool for in vivo preclinical studies. To detect brain three-dimensional (3D) microstructures, we improved the ex vivo MEMRI method for mouse brains after in vivo Mn administration and obtained high-resolution MRIs using a cryogenic radiofrequency (RF) coil. Male C57BL/6 mice (n = 8) were injected with 50 mM MnCl2 intravenously and MEMRIs of the brain were acquired in vivo after 24 h, followed by perfusion fixation with a 4% paraformaldehyde (PFA) solution. High-resolution 25-μm isotropic MRIs were successfully acquired from the extracted brain tissue and could identify the brain microstructures, especially in the hippocampus [the pyramidal cell layer through CA1–3 and the dentate gyrus (DG) granular layers (GLs)], cell layers of cerebellum, three sub-regions of the deep cerebellar nucleus, and white matter (WM) structures [e.g., the fasciculus retroflexus (fr) and optic tract in the thalamus]. The following technical conditions were also examined: (i) the longitudinal stability of Mn-enhanced ex vivo tissue after in vivo administration; and (ii) the effects of mixing glutaraldehyde (GA) with the fixative solution for the preservation of in vivo MEMRI contrast. Our results indicate that ex vivo MEMRI observations made shortly after fixation maintain the contrast observed in vivo. This research will be useful for non-destructive whole-brain pathological analysis.

The anatomical pathway from the mesodiencephalic junction to the inferior olive relays perioral sensory signals to the cerebellum in the mouse.

Key points Perioral tactile signals are transmitted via the infraorbital nerve (ION) to trigeminal nuclei. Each cerebellar Purkinje cell (PC) receives this signal as complex spikes (CSs) via a climbing fibre (CF) emerging from the inferior olive (IO).The anatomical pathway from trigeminal nuclei to the IO is not clearly identified.In the present study, we examined candidate anatomical pathways for perioral sensory signalling by analysing CSs recorded from PCs in male mice by single unit recording.CS generation by ION stimulation was inhibited by injection of a GABAA receptor agonist, muscimol, into the contralateral mesodiencephalic junction, which is referred to as the area parafascicularis prerubralis (PfPr). The number of CSs evoked by mechanical whisker stimulation was also decreased by contralateral PfPr inhibition.These results suggest the existence of a sensory signalling pathway to the IO via the PfPr in mice. Perioral tactile signals are transmitted via the infraorbital nerve (ION) to trigeminal nuclei. Each cerebellar Purkinje cell receives this signal as complex spikes (CSs) via a climbing fibre emerging from the inferior olive (IO). However, the anatomical pathway from the trigeminal nuclei to the IO is not clearly identified. In the present study, we recorded CSs from Purkinje cells in male mice by single unit recording, and examined the signal transduction pathway. CSs were evoked by electrical stimulation of the ipsilateral or contralateral ION with a latency of 20–70 ms. CS generation by ipsilateral ION stimulation was inhibited by injection of a GABAA receptor agonist, muscimol, into the contralateral mesodiencephalic junction, ranging from around the fasciculus retroflexus to the interstitial nucleus of Cajal, which is referred to as the area parafascicularis prerubralis (PfPr). CSs evoked by contralateral ION stimulation were also suppressed by muscimol injection into the PfPr, although the effective area was more restricted. Furthermore, CSs evoked by mechanical stimulation around the whisker region were suppressed by PfPr inhibition. We also found that the primary motor cortex plays a role to suppress this signalling pathway. These results indicate the existence of an anatomical pathway for conducting perioral sensory signals to the IO via the PfPr.