Monique Touret

The raphe nuclei of the cat brain stem: A topographical atlas of their efferent projections as revealed by autoradiography

Stereotaxic injections of [14C]leucine were made in nulei raphe centralis superior, raphe dorsalis, raphe magnus and raphe pontis of the cat. The organization of the regional connections was outlined in a stereotaxic atlas using the autoradiographic tracing method: the majority of the ascending pathways from the rostral raphe nuclei are directed mainly through a ventrolateral bundle via the ventral tegmental area of Tsai, with some lateral extensions to the substantia nigra, and then through the fields of Forel and the zona incerta. More rostrally the fibers are joined to the medial forebrain bundle through the hypothalamic region up to the preoptic area or the diagonal band of Broca. Multiple divisions leave this tract towards the epithalamic or the intralaminar thalamic nuclei, the stria terminalis, the septum, the capsula interna and the ansa lenticularis. The bulk of the rostral projections terminates in the frontal lobe, while some labeling is scarcely distributed throughout the rest of the neocortex. The projections of nucleus (n.) raphe centralis superior are specifically associated with the n. interpeduncularis, the mammillary bodies and the hippocampal formation while the n. raphe dorsalis innervates selectively the lateral geniculate bodies, striatus, piriform lobes, olfactory bulb and amygdala. The rest of the ascending fibers form the centrolateral or the dorsal ascending tracts radiating either in the reticular mesencephalic formation or in the periventricular gray matter. On the contrary there are heavy descending projections from n. raphe centralis superior which distribute to the main nuclei of the brain stem, the central gray matter and the cerebellum. The ascending projections form the caudal raphe nuclei are much less dense. They disseminate mainly in the colliculus superior, the pretectum, the nucleus of the posterior commissure, the preoculomotor complex and the intralaminar nuclei of the thalamus. From n. raphe pontis, a dense labeling is selectively localized at the n. paraventricularis hypothalami with some rostral extensions to limbic areas. Diffuse caudal and rostral projections from both nuclei are observed in the mesencephalic, pontobulbar reticular formation and the cerebellum. The main differences come from the specific localization of their descending bulbospinal tracts inside the lateroventral funiculus of the spinal cervical cord.

Afferent projections to the cat locus coeruleus as visualized by the horseradish peroxidase technique

Summary Using a recently developed retrograde tracer technique with horseradish peroxidase (HRP), attempts were made to identify afferent projections to the dorso-lateral part of the pontomesencephalic tegmental areas, including the nucleus locus coeruleus (LC), locus subcoeruleus (LSC), parabrachialis lateralis (Pbl), Kolliker-Fuse (K-F), reticularis pontis oralis (RPO), reticularis pontis caudalis (RPC), as well as an area rostral to the Pbl and dorsolateral to the brachium conjunctivum (mesencephalic reticular formation (MRF) area). It was revealed that the nucleus raphe dorsalis projects widely to all the studied pontomesencephalic tegmental nuclei. In addition, the LC was found to project to all the contralateral pontine tegmental structures which were studied. Following the injection of HRP into the dorsomedial part of the LC (‘principal LC’ or ‘LC’), HRP labeled neurons were observed almost restricted to the nucleus raphe dorsalis. In contrast, following the injection into the ventrolateral part of the LC (LCα) or into other tegmental areas, the HRP containing neurons were observed widely distributed in the brain, extending from the diencephalon to the rhombencephalon. Especially in the case where injection was made into the LCα, numerous HRP positive cells appeared in the nucleus raphe pontis, magnus and substantia nigra, and were also identified in other brain structures, the topography of which corresponded to that of the catecholamine-containing neurons of the rat (group A1–A14). The present results confirm some previous reports on the afferent connections of the LC described in the rat, cat and rabbit, and further indicate the richness of afferent projections to the dorsolateral pontine tegmental areas. The present study also shows the heterogeneity existing between the main LC areas and the subcoeruleus areas, as well as between the dorsomedial part of the main LC (principal LC) and the ventrolateral part of this nucleus (LCα).

Spinal projections from the lower brain stem in the cat as demonstrated by the horseradish peroxidase technique. I. Origins of the reticulospinal tracts and their funicular trajectories

Using a retrograde tracer technique with horseradish peroxidase (HRP) attempts were made to determine the origins of reticulospinal tracts and their funicular trajectories. Reticulospinal tracts originating from the mesencephalic reticular formation (RF) were composed of: (1) descending projections arising from the cluster of cells located just lateral to the periaqueductal gray that course in the anterior funiculus (AF) and ventral part of the lateral funiculus (LF) with ipsilateral predominance; and (2) projections from the cluster of cells located dorsal to the brachium conjunctivum that course in the ipsilateral LF. Origins of the pontine reticulospinal tracts arising from the n. reticularis pontis oralis (Poo) have been divided qnto three parts: (1) medial one-third; (2) middle; and (3) ventrolateral. The axons from the medial part descend ipsilaterally via the medial part of the AF, while the axons from the ventrolateral part of the Poo give rise to diffuse descending projections in the AF and LF. The middle part of the Poo has been further subdivided into: (1) dorsal part that gives rise to spinal projections ipsilaterally in the ventrolateral funiculus (VLF); and (2) ventral, particularly its upper part, whose axons descend bilaterally via the DLF. Origins of reticulospinal tracts from the n. reticularis pontis caudalis (Poc) could be divided into three parts: (1) medial; (2) dorsolateral; and (3) ventrolateral. The medial part of the Poc is a source of axons via the medial part of the ipsilateral AF, while the ventrolateral part of the nucleus is a source of axons via the contralateral LF. The spinal projections from the dorsolateral part of the Poc appears to course diffusely in the AF and LF, but with DLF predominance. The n. reticularis gigantocellularis (Gc) was found to be a main medullary source of the spinal projections in the ipsilateral AF, while n. reticularis magnocellularis (Mc) is the major source of the fibers coursing ipsilaterally in the VLF. The most medial part of the Mc descends ipsilaterally via the medial part of the AF, while the ventrolateral part of the nucleus together with the n. reticularis lateralis of Meesen and Olszewski descends ipsilaterally via the DLF. It has also been found that the axons from the n. reticularis paramedianus pass via both the AF and LF with ipsilateral predominance, while the n. reticularis dorsalis and ventralis course via the LF with ipsilateral predominance.

Afferent connections of the nucleus raphe dorsalis in the cat as visualized by the horseradish peroxidase technique.

Using a retrograde tracer technique with protein horseradish peroxidase (HRP), attempts were made to determine afferent projections to the nucleus raphe dorsalis (NRD). As a control, the injection of the HRP was also made into one of the following structures adjacent to the NRD: (1) mesencephalic periaque ductal gray; (2) nucleus linearis intermedius; and (3) third cranial nucleus. The present results indicate that the NRD, particularly is rostral part, receives direct projections arising from: (1) locus coeruleus complex (locus coeruleus, locus coerulus alpha, and locus subcoeruleus); (2) parabrachial nuclei (nucleus parabrachialis lateralis and medialis); (3) nucleus laterodorsalis tegmenti; (4) griseum centrale pontis, particularly the caudal part of the nucleus incertus; (5) substantia nigra; (6) lateral habenular nucleus; (7) hypothalamic areas, particularly dorsal and lateral hypothalamic areas; (8) preoptic areas; (9) anarea dorso-lateral to the inferior olivary complex and medial to the lateral reticular nucleus; and (10) raphe nuclei; particularly nucleus linearis intermedius, centralis superior, pontis and magnus. The present findings thus confirm some previous reports on the afferent projections to the NRD described in the cat and rat, and further indicate the richness of afferent connections of the NRD. Some problems of the peroxidase technique have also been discussed.

Tegmentoreticular projections with special reference to the muscular atonia during paradoxical sleep in the cat: An HRP study

Using the retrograde tracer technique with horseradish peroxidase (HRP), attempts were made to determine the cells of origin and the descending pathway of the tegmentoreticular projections in order to give an anatomical substrate for the physiological phenomenon of the postural atonia observed during paradoxical sleep (PS) in the cat. The HRP was injected into various parts of the pontomedullary reticular formation (RF) including the caudal raphe nuclei, nucleus (n.) reticularis gigantocellularis (Gc), n. reticularis magnocellularis (Mc), and other pontomedullary structures adjacent to the Mc. The results indicated that the HRP injection into the Mc, particularly its caudal and lateral two-thirds, resulted in specific labeling of cells located in an area just medial to the LCa together with those in the most medial part of the LCa. Bilateral lesions of these pontine structures have been reported to suppress the atonia otherwise observed during PS in the normal cat. In addition to the HRP labeled cells, we have also observed HRP filled fiber bundles directed to labeled cells in the medial part of the LCa and immediately adjacent tegmental RF area. The same course of HRP labeled fiber bundles was also observed together with HRP labeled cells in the Mc after HRP injections into the medial part of the LCa area, indicating the existence of an interconnection between the LCa area and the Mc. The location of the tegmentoreticular pathway corresponded to that of the lesions effective to suppress the muscular atonia during PS. HRP injections into the caudal medullar caudal to the Mc, on the other hand, resulted in no or almost no HRP labeled cells in the area medial to the LCa, in spite of the presence of HRP containing neurons in other parts of the pontomedullary RF areas, showing that the tegmentoreticular projections as described above terminate almost exclusively in the Mc.

Spinal projections from the lower brain stem in the cat as demonstrated by the horseradish peroxidase technique. II. projections from the dorsolateral pontine tegmentum and raphe nuclei

The descending projections to the spinal cord arising from the dorsolateral pontine tegmentum and brain stem raphe nuclei have been investigated by means of the horseradish peroxidase (HRP) technique. Particular attention was taken to clarify the cells of origin and the funicular trajectory of these spinal projections. After injections of HRP into the spinal cord, a significant of HRP labeled neurons were observed in the following dorsolateral pontine tegmental structures: (1) an area ventral to the nucleus cuneiformis; (2) principal locus coeruleus; (3) locus coeruleus a; (4) locuse subcoeruleus; (5) Kölliker-Fuse nucleus; and (6) nucleus parabrachialis lateralis. As a rule, the projections are ipsilateral and descendaphe-spinal projections, we have demonstrated that the nucleus raphe dorsalis also sends axons to the cervical segment of the spinal cord. Furthermore, in accord with previous reports, HRP labeled cells were also identified in the nucleus raphe magnus, pallidus and obscurus, but not in the nucleus raphe centralis superior and pontis. On the whole the present study further clarified the organization of spinal projections from the dorsolateral pons and raphe nuclei and provided some additional anatomical data for the physiology of the tegmentospinal and raphe-spinal projections.

Oligodendrocytes are damaged by neuromyelitis optica immunoglobulin G via astrocyte injury

Devics neuromyelitis optica is an inflammatory demyelinating disorder normally restricted to the optic nerves and spinal cord. Since the identification of a specific autoantibody directed against aquaporin 4, neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody, neuromyelitis optica has been considered an entity distinct from multiple sclerosis. Recent findings indicate that the neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody has a pathogenic role through complement-dependent astrocyte toxicity. However, the link with demyelination remains elusive. Autoantibodies can act as receptor agonists/antagonists or alter antigen density in their target cells. We hypothesized that the neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody impairs astrocytic function and secondarily leads to demyelination. Rat astrocytes and oligodendrocytes from primary cultures and rat optic nerves were exposed long-term (24 h) to immunoglobulin G in the absence of complement. Immunoglobulin G was purified from the serum of patients with neuromyelitis optica who were either neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody positive or negative, as well as from healthy controls. Flow cytometry analysis showed a reduction of membrane aquaporin 4 and glutamate transporter type 1 on astrocytes following contact with immunoglobulin G purified from neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody positive serum only. The activity of glutamine synthetase, an astrocyte enzyme converting glutamate into glutamine, decreased in parallel, indicating astrocyte dysfunction. Treatment also reduced oligodendrocytic cell processes and approximately 30% oligodendrocytes died. This deleterious effect was confirmed ex vivo; exposed optic nerves showed reduction of myelin basic protein. Immunoglobulin G from neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody seronegative patients and from healthy controls had no similar effect. Neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody did not directly injure oligodendrocytes cultured without astrocytes. A toxic bystander effect of astrocytes damaged by neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody on oligodendrocytes was identified. Progressive accumulation of glutamate in the culture medium of neuromyelitis optica-immunoglobulin G/aquaporin 4-antibody-treated glial cells supported the hypothesis of a glutamate-mediated excitotoxic death of oligodendrocytes in our models. Moreover, co-treatment of glial cultures with neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody and d+2-amino-5-phosphonopentanoic acid, a competitive antagonist at the N-methyl-d-aspartate/glutamate receptor, partially protected oligodendrocytes. Co-immunolabelling of oligodendrocyte markers and neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody showed that astrocytic positive processes were in close contact with oligodendrocytes and myelin in rat optic nerves and spinal cord, but far less so in other parts of the central nervous system. This suggests a bystander effect of neuromyelitis optica-immunoglobulin G-damaged astrocytes on oligodendrocytes in the nervous tissues affected by neuromyelitis optica. In conclusion, in these cell culture models we found a direct, complement-independent effect of neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody on astrocytes, with secondary damage to oligodendrocytes possibly resulting from glutamate-mediated excitotoxicity. These mechanisms could add to the complement-induced damage, particularly the demyelination, seen in vivo.

Cerebrospinal fluid dendritic cells infiltrate the brain parenchyma and target the cervical lymph nodes under neuroinflammatory conditions.

Background In many neuroinflammatory diseases, dendritic cells (DCs) accumulate in several compartments of the central nervous system (CNS), including the cerebrospinal fluid (CSF). Myeloid DCs invading the inflamed CNS are thus thought to play a major role in the initiation and perpetuation of CNS-targeted autoimmune responses. We previously reported that, in normal rats, DCs injected intra-CSF migrated outside the CNS and reached the B-cell zone of cervical lymph nodes. However, there is yet no information on the migratory behavior of CSF-circulating DCs under neuroinflammatory conditions. Methodology/Principal Findings To address this issue, we performed in vivo transfer experiments in rats suffering from experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. EAE or control rats were injected intra-CSF with bone marrow-derived myeloid DCs labeled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFSE). In parallel experiments, fluorescent microspheres were injected intra-CSF to EAE rats in order to track endogenous antigen-presenting cells (APCs). Animals were then sacrificed on day 1 or 8 post-injection and their brain and peripheral lymph nodes were assessed for the presence of microspheres+ APCs or CFSE+ DCs by immunohistology and/or FACS analysis. Data showed that in EAE rats, DCs injected intra-CSF substantially infiltrated several compartments of the inflamed CNS, including the periventricular demyelinating lesions. We also found that in EAE rats, as compared to controls, a larger number of intra-CSF injected DCs reached the cervical lymph nodes. This migratory behavior was accompanied by an accentuation of EAE clinical signs and an increased systemic antibody response against myelin oligodendrocyte glycoprotein, a major immunogenic myelin antigen. Conclusions/Significance Altogether, these results indicate that CSF-circulating DCs are able to both survey the inflamed brain and to reach the cervical lymph nodes. In EAE and maybe multiple sclerosis, CSF-circulating DCs may thus support the immune responses that develop within and outside the inflamed CNS.

Decreased expression of glutamate transporters in genetic absence epilepsy rats before seizure occurrence

In absence epilepsy, epileptogenic processes are suspected of involving an imbalance between GABAergic inhibition and glutamatergic excitation. Here, we describe alteration of the expression of glutamate transporters in rats with genetic absence (the Genetic Absence Epilepsy Rats from Strasbourg: GAERS). In these rats, epileptic discharges, recorded in the thalamo‐cortical network, appear around 40 days after birth. In adult rats no alteration of the protein expression of the glutamate transporters was observed. In 30‐day‐old GAERS protein levels (quantified by western blot) were lower in the cortex by 21% and 35% for the glial transporters GLT1 and GLAST, respectively, and by 32% for the neuronal transporter EAAC1 in the thalamus compared to control rats. In addition, the expression and activity of GLAST were decreased by 50% in newborn GAERS cortical astrocytes grown in primary culture. The lack of modification of the protein levels of glutamatergic transporters in adult epileptic GAERS, in spite of mRNA variations (quantified by RT‐PCR), suggests that they are not involved in the pathogeny of spike‐and‐wave discharges. In contrast, the alteration of glutamate transporter expression, observed before the establishment of epileptic discharges, could reflect an abnormal maturation of the glutamatergic neurone–glia circuitry.

Cortical glutamate metabolism is enhanced in a genetic model of absence epilepsy

Disturbances in GABAergic and glutamatergic neurotransmission in the thalamocortical loop are involved in absence seizures. Here, we examined potential disturbances in metabolism and interactions between neurons and glia in 5-month-old genetic absence epilepsy rats from Strasbourg (GAERS) and nonepileptic rats (NER). Animals received [1-13C]glucose and [1,2-13C]acetate, the preferential substrates of neurons and astrocytes, respectively. Extracts from cerebral cortex, thalamus, and hippocampus were analyzed by 13C nuclear magnetic resonance spectroscopy. Most changes were detected in the cortex. Pyruvate metabolism was enhanced as evidenced by increases of lactate, and labeled and unlabeled alanine. Neuronal mitochondrial metabolism was also enhanced as detected by elevated amounts of N-acetylaspartate and nicotinamide adenine dinucleotide as well as increased incorporation of label from [2-13C]acetyl CoA into glutamate, glutamine, and aspartate. Likewise, mitochondrial metabolism in astrocytes was increased. Changes in thalamus were restricted to increased concentration and labeling of glutamine. Changes in the hippocampus were similar to those in the cortex. This increase in glutamate-glutamine metabolism in cortical neurons and astrocytes accompanied by a decreased gamma aminobyturic acid level may lead to impaired thalamic filter function. Hence, reduced sensory input to cortex could allow the occurrence of spike-and-wave discharges in the thalamocortical loop. Increased glutamatergic output from the cortex to hippocampus may be the underlying cause of improved learning in GAERS.