Distribution of Glutamatergic Receptors and GAD mRNA-Containing Neurons in the Vestibular Nuclei of Normal and Hemilabyrinthectomized Rats

Vestibular compensation is an attractive model for investigations of cellular mechanisms underlying post-lesional plasticity in the adult central nervous system. Immediately after hemilabyrinthectomy, the spontaneous activity in the deafferented second-order vestibular neurons falls to zero, resulting in a strong asymmetry between the resting discharge of the vestibular complexes on the lesioned and intact sides. This asymmetry most probably causes the static and dynamic vestibular deficits observed in the acute stage. After ～50 h, the deafferented vestibular neurons recover a quasi-normal resting activity which is thought to be the key of the compensation of the static vestibular syndromes. However, the molecular mechanisms underlying this recovery are unknown. In this study, we investigate possible changes in the distribution of glutamatergic N-methyl-d-aspartate (NMDA) and glutamate metabotropic receptors and of glutamate decarboxylase 67k (GAD 67k) mRNAs in the deafferented vestibular neurons induced by the labyrinthine lesion. Specific radioactive oligonucleotides were used to probe sections of rat vestibular nuclei according to in situ hybridization methods. Animals were killed at different times (5 h, 3 days and 3 weeks) following the lesion. Signal was detected by means of film or emulsion autoradiography. In the normal animals, several brainstem regions including the medial, lateral, inferior and superior vestibular nuclei were densely labelled by the antisense oligonucleotide NMDAR1 probe. However, the vestibular nuclei were not labelled by the glutamate metabotropic oligonucleotide antisense probe (mGluR 1). The GAD 67k antisense oligonucleotide probe labelled numerous small- to medium-sized central vestibular neurons but not the larger cell bodies in the lateral vestibular nucleus. This agrees with previous studies. In the hemilabyrinthectomized rats, no asymmetry could be detected, at either the autoradiographic or cellular levels, between the two medial vestibular nuclei whatever the probe used and whatever the delay following the lesion. However, for the NMDAR1 probe, the mean density of silver grains in both the deafferented and intact medial vestibular neurons was 20% lower 5 h after the lesion. Three days and 3 weeks later, the intensity of labelling over all cells was the same as in the control group. Further studies are necessary to confirm the relatively weak modification of the NMDAR1 mRNAs expression and to exclude a change of GAD 65 and of other NMDA subunit mRNAs during the vestibular compensation process.     

Development of the brain stem in the rat. III. Thymidine-radiographic study of the time of origin of neurons of the vestibular and auditory nuclei of the upper medulla

Groups of pregnant rats were injected with two successive daily doses of ³H-thymidine form gestational days 12 and 13 (E12 + 13) until the day before parturition (E21 + 22). In adult progeny of the injected rats the proportion of neurons generated on specific embryonic days was determined quantitatively in the vestibular and auditory nuclei of the upper medulla. In the vestibular nuclei, neurons are generated between days E11 and E15 in an overlapping sequential order, yielding a lateral-to-medial and a rostral-to-caudal internuclear gradient. In the lateral vestibular nucleus peak production time is day E12; in the superior nucleus, E13; in the inferior nucleus, E13 and E14; and in the medial nucleus, E14. The early generation of neurons of the lateral vestibular nucleus may reflect the early differentiation of the circuit from the gravity receptors (utricle) to neurons of the spinal cord controlling postural balance. The later production of neurons of the superior vestibular nucleus may reflect the subsequent differentiation of the circuit from the rotational receptors (semicircular canals) to the neurons of the brain stem controlling eye movements. The generation time of neurons of the nucleus prepositus hypoglossi overlaps with that of the medial vestibular nucleus. The neurons of the anteroventral and posteroventral cochlear nuclei are produced form days E13 to E17, with no temporal differences between the two nuclei. The neurons of the dorsal cochlear nucleus are generated over a very long time span, beginning on day E12 and extending into the postnatal period. There is a sequence in the production of neurons forming the different layers of the dorsal cochlear nucleus in the following order: pyramidal cells, cells of the inner layer, cells of the outer layer and, finally, cells of the granular layer. There is also a sequential production of neurons in four nuclei of the superior olivary complex. In the lateral trapezoid nucleus peak production time is day E12; in the medial superior olivary nucleus, day E13; in the medial trapezoid nucleus, day E15; and in the lateral superior olivary nucleus, day E16. This order yields a medial-to-lateral gradient in the dorsal aspect of the superior olivary complex, and a lateral-to-medial gradient ventrally. These mirror-image gradients were also seen intranuclearly in the lateral superior olivary nucleus and the medial trapezoid nucleus. The cytogenetic gradients could not be related to tonotopic representation; however, they could be related to the lateral location of ipsilateral cochlear nucleus input to the lateral superior olivary nucleus and the medial location of the contralateral cochlear nucleus input to the medial trapezoid nucleus.     

Long-term interaction between microglial cells and cochlear nucleus neurons after bilateral cochlear ablation

The removal of afferent activity has been reported to modify neuronal activity in the cochlear nucleus of adult rats. After cell damage, microglial cells are rapidly activated, initiating a series of cellular responses that influences neuronal function and survival. To investigate how this glial response occurs and how it might influence injured neurons, bilateral cochlear ablations were performed on adult rats to examine the short-term (16 and 24 hours and 4 and 7 days) and long-term (15, 30, and 100 days) changes in the distribution and morphology of microglial cells (immunostained with the ionized calcium-binding adaptor molecule 1; Iba-1) and the interaction of microglial cells with deafferented neurons in the ventral cochlear nucleus. A significant increase in the mean cross-sectional area and Iba-1 immunostaining of microglial cells in the cochlear nucleus was observed at all survival times after the ablation compared with control animals. These increases were concomitant with an increase in the area of Iba-1 immunostaining at 24 hours and 4, 7, and 15 days postablation. Additionally, microglial cells were frequently seen apposing the cell bodies and dendrites of auditory neurons at 7, 15, and 30 days postablation. In summary, these results provide evidence for persistent glial activation in the ventral cochlear nucleus and suggest that long-term interaction occurs between microglial cells and deafferented cochlear nucleus neurons following bilateral cochlear ablation, which could facilitate the remodeling of the affected neuronal circuits.     

Alterations in calretinin immunostaining in the ferret superior olivary complex after cochlear ablation

In this study, we used image analysis to assess changes in calretinin immunoreactivity in the lateral (LSO) and medial (MSO) superior olivary nuclei in ferrets 2 months after unilateral cochlear ablations at 30-40 days of age, soon after hearing onset. These two nuclei are the first significant sites of binaural convergence in the ascending auditory system, and both receive direct projections from the deafferented cochlear nucleus. Cochlear ablation results in a decrease in the overall level of calretinin immunostaining within the LSO ipsilaterally compared with the contralateral side and with control animals and within the MSO bilaterally compared with control ferrets. In addition, the level of calretinin immunostaining ipsilaterally within neurons in the LSO was significantly less in cochlear ablated than control animals. In contrast, there was no effect of cochlear ablation on the level of calretinin immunostaining within neurons either in the contralateral LSO or in the MSO. These results are consistent with a downregulation in calretinin within the neuropil of MSO bilaterally and LSO ipsilaterally, as well as a downregulation in calretinin within somata in the ipsilateral LSO as a result of unilateral cochlear ablation soon after hearing onset. Thus, cochlear-driven activity appears to affect calcium binding protein levels in both neuropil and neurons within the superior olivary complex.     

New neurons in the vestibular nuclei complex after unilateral vestibular neurectomy in the adult cat

Recent findings revealed a reactive neurogenesis after lesions and in several models of disease. After unilateral vestibular neurectomy (UVN), we previously reported gamma-aminobutyric acid (GABA)ergic neurons are upregulated in the vestibular nuclei (VN) in the adult cat. Here, we ask whether this upregulation of GABAergic neurons resulted from a reactive neurogenesis. To determine the time course of cell proliferation in response to UVN, 5-bromo-2'-deoxyuridine (BrdU) was injected 3 h, 1, 3, 7, 15 and 30 days after UVN. We investigated the survival and differentiation in UVN cats injected with BrdU at 3 days and perfused 30 days after UVN. Results show a high number of BrdU-immunoreactive nuclei in the deafferented VN with a peak at 3 days after UVN and a decrease at 30 days. Most of the newly generated cells survived up to 1 month after UVN and gave rise to a variety of cell types. Confocal analysis revealed three cell lineages: microglial cells (OX 42/BrdU-immunoreactive cells); astrocytes [glial fibrillary acidic protein (GFAP)/BrdU-immunoreactive cells]; and neurons (NeuN/BrdU-immunoreactive cells). That UVN induced new neurons was confirmed by an additional marker (nestin) expressed by neural precursor cells. We show that most of the newly generated neurons have a GABAergic phenotype [glutamate decarboxylase (GAD)-67/BrdU-immunoreactive cells]. Morphological analysis showed two subtypes of GABAergic neurons: medium and small (30 vs. 10 microm, respectively). This is the first report of reactive neurogenesis in the deafferented VN in the adult mammalian CNS.     

Modification of tenascin-R expression following unilateral labyrinthectomy in rats indicates its possible role in neural plasticity of the vestibular neural circuit

We have previously found that unilateral labyrinthectomy is accompanied by modification of hyaluronan and chondroitin sulfate proteoglycan staining in the lateral vestibular nucleus of rats and the time course of subsequent reorganization of extracellular matrix assembly correlates to the restoration of impaired vestibular function. The tenascin-R has repelling effect on pathfinding during axonal growth/regrowth, and thus inhibits neural circuit repair. By using immunohistochemical method, we studied the modification of tenascin-R expression in the superior, medial, lateral, and descending vestibular nuclei of the rat following unilateral labyrinthectomy. On postoperative day 1, tenascin-R reaction in the perineuronal nets disappeared on the side of labyrinthectomy in the superior, lateral, medial, and rostral part of the descending vestibular nuclei. On survival day 3, the staining intensity of tenascin-R reaction in perineuronal nets recovered on the operated side of the medial vestibular nucleus, whereas it was restored by the time of postoperative day 7 in the superior, lateral and rostral part of the descending vestibular nuclei. The staining intensity of tenascin-R reaction remained unchanged in the caudal part of the descending vestibular nucleus bilaterally. Regional differences in the modification of tenascin-R expression presented here may be associated with different roles of individual vestibular nuclei in the compensatory processes. The decreased expression of the tenascin-R may suggest the extracellular facilitation of plastic modifications in the vestibular neural circuit after lesion of the labyrinthine receptors.     

Basic fibroblast growth factor expression and tenascin C immunoreactivity after partial unilateral hemitransection of the rat brain

Basic fibroblast growth factor (bFGF) gene expression as well as its immunoreactivity were studied after partial unilateral hemitransection of the rat brain during a time course of 24 h, 72 h, 7 and 14 days. The mechanical injury resulted in a global increase of bFGF gene expression at the 24-h time interval. This global increase was seen at the ipsilateral site at the level of the lesion as well as rostral to the lesion in the ipsilateral hemisphere. The upregulation in bFGF gene expression was in most of the areas investigated due to an upregulation in glial cells as seen by means of nonradioactive in situ hybridization compared with immunocytochemistry for glial fibrillary acidic protein (GFAP). Basic FGF immunoreactivity (IR) was increased around the lesion in glial cell nuclei 7 days after the injury. This increase was also detected in GFAP positive glial cells surrounding small vessels in the lesioned area. Moreover, in the present paper we demonstrate increased tenascin immunoreactivity in the lesioned area 7 days after injury. The tenascin IR was increased at the edges of the lesion as well as in vessel like structures. The tenascin IR was partially codistributed with GFAP IR in the lesioned area. The lesion was also characterized by an increase in vimentin IR as well as in laminin IR. It is suggested that the observed changes in the expression of bFGF, matrix proteins (laminin, tenascin) and intermediate filaments (vimentin) are involved in (a) tissue repair, (b) protection of neuronal cells from excitotoxic influences and (c) formation of new vessels in the lesioned area.     

Stimulation by cochlear implant in unilaterally deaf rats reverses the decrease of inhibitory transmission in the inferior colliculus

In the last decade, numerous studies have investigated synaptic transmission changes in various auditory nuclei after unilateral cochlear injury. However, few data are available concerning the potential effect of electrical stimulation of the deafferented auditory nerve on the inhibitory neurotransmission in these nuclei. We report here for the first time the effect of chronic electrical stimulation of the deafferented auditory nerve on alpha1 subunit of the glycinergic receptor (GlyRalpha1) and glutamic acid decarboxylase (GAD)67 expression in the central nucleus of inferior colliculus (CIC). Adult rats were unilaterally cochleectomized by intracochlear neomycin sulphate injection. Fifteen days later, the ipsilateral auditory nerve was chronically stimulated either 4, 8 or 22 h daily, for 5 days using intracochlear bipolar electrodes. GlyRalpha1 and GAD67 mRNA and protein were quantified in the CIC using in situ hybridization and immunohistofluorescence methods. Our data showed that as after surgical ablation, GlyRalpha1 and GAD67 expression were strongly decreased in the contralateral CIC after unilateral chemical cochleectomy. Most importantly, these postlesional down-modulations were significantly reversed by chronic electrical stimulation of the deafferented auditory nerve. This recovery, however, did not persist for more than 5 days after the cessation of the deafferented auditory nerve electrical stimulation. Thus, downregulations of GlyRalpha1 and GAD67 may be involved both in the increased excitability observed in the CIC after unilateral deafness and consequently in the tinnitus frequently observed in unilateral adult deaf patients. Electrical stimulation of the deafferented auditory nerve in patients may be a potential new approach for treating tinnitus with unilateral hearing loss.     

Electrical stimulation of the cochlear nerve in rats: analysis of c-Fos expression in auditory brainstem nuclei

We investigated functional activation of central auditory brainstem nuclei in response to direct electrical stimulation of the cochlear nerve using c-Fos immunoreactivity as a marker for functional mapping. The cochlear nerve was stimulated in the cerebellopontine angle of Lewis rats applying biphasic electrical pulses (120-250 muA, 5 Hz) for 30 min. In a control group, bilateral cochlectomy was performed in order to assess the basal expression of c-Fos in the auditory brainstem nuclei. The completeness of cochlear ablations and the response of auditory brainstem nuclei to electrical stimulation were electrophysiologically verified. C-Fos immunohistochemistry was performed using the free floating method. In anaesthetized animals with unilateral electrical stimulation of the cochlear nerve, increased expression of c-Fos was detected in the ipsilateral ventral cochlear nucleus (VCN), in the dorsal cochlear nucleus bilaterally (DCN), in the ipsilateral lateral superior olive (LSO) and in the contralateral inferior colliculus (IC). A bilateral slight increase of c-Fos expression in all subdivisions of the lateral lemniscus (LL) did not reach statistical significance. Contralateral inhibition of the nuclei of the trapezoid body (TB) was observed. Our data show that unilateral electrical stimulation of the cochlear nerve leads to increased expression of c-Fos in most auditory brainstem nuclei, similar to monaural auditory stimulation. They also confirm previous studies suggesting inhibitory connections between the cochlear nuclei. C-Fos immunoreactivity mapping is an efficient tool to detect functional changes following direct electrical stimulation of the cochlear nerve on the cellular level. This could be particularly helpful in studies of differential activation of the central auditory system by experimental cochlear and brainstem implants.     

Increased expression of GABA transporters, GAT-1 and GAT-3, in the deafferented superior colliculus of the rat

GABA transporters (GATs) play a critical role in the translemmal transport of GABA in neurons and glial cells. Two major brain GATs, GAT-1 and GAT-3, are found in astrocytes in the adult brain. Astroglia demonstrate morphological and molecular changes in response to brain injury and deafferentation. The present study was designed to determine whether the expression of GATs changes after nerve deafferentation using the rat superior colliculus (SC) as a model. The immunoreactivity for GAT-1 and GAT-3, as well as GABA and glutamic acid decarboxylase (GAD)-65 and GAD-67, was studied in the SC of control rats and rats with unilateral optic nerve transections. Immunolabeling for both GAT-1 and GAT-3 was increased in the neuropil of the denervated SC as compared to that for the SC of control rats or for the unaffected SC of experimental rats. In contrast, immunoreactivity for GABA, GAD-65 and GAD-67 was not altered. The change in the immunolabeling of GAT-1 and GAT-3 was detectable at 1 day postlesion and became more evident between 3–30 days postlesion. At the electron microscopic level, immunoreactivity for both GAT-1 and GAT-3 in the unaffected SC was localized to astrocytic processes, whereas GAT-1 immunolabeling was also present in synaptic terminals. In the deafferented SC, immunolabeling for both GATs was elevated in the somata and processes of hypertrophied astrocytes as compared to that in the unaffected SC, whereas GAT-1 labeling in neuronal profiles was largely unchanged. A substantial increase of GAT-1 and GAT-3 in astrocytes following optic nerve transection suggests that these cells play a role in modulating GABA's action in the deafferented SC.     

Vimentin immunoreactive glial cells in the fish optic nerve: Implications for regeneration

The poor regenerative ability of neurons of the central nervous system in mammals, as compared with their counterpart in fish or amphibians, is thought to stem from differences in their immediate nonneuronal environment and its response to axonal injury. We describe one aspect of the environmental response to axonal injury in a spontaneously regonerating system-the fish optic nerve. The aspect under investigation was the reaction of glial cells at the injury site. This was examined by the use of antibodies that specifically recognize vimentin in fish glial cells. In the present study, affinity-purified vimentin antibodies were raised against a nonconserved N-terminal 14-amino acid peptide, which was predicted from the nucleotide sequence of vimentin. These antibodies were found to react specifically with glial cells in vitro. Moreover, the antivimentin antibodies stained both the optic nerve and the optic tract, but with different patterns. Specificity of the antibodies was verified by protein immunoblotting, tissue distribution, and labeling patterns. After injury, vimentin immunoreactivity initially disappeared from the site of the lesion due to cell death. Early signs of glial cell migration toward the injury site were evident a few days later. It is suggested that the reappearance of vimentin-positive glial cells at the site of injury is associated with axonal elongation across it, and that they contribute to the regenerative ability of the fish optic nerve. © 1994 Wiley-Liss, Inc.     

Organization of radial and non-radial glia in the developing rat thalamus

The organization of glia and its relationship with migrating neurons were studied in the rat developing thalamus with immunocytochemistry by using light, confocal, and electron microscopy. Carbocyanine labeling in cultured slice of the embryonic diencephalon was also used. At embryonic day (E) 14, vimentin immunoreactivity was observed in radial fascicles spanning the neuroepithelium and extending from the ventricular zone to the lateral surface of the diencephalic vesicle. Vimentin-immunopositive fibers orthogonal to the radial ones were also detected at subsequent developmental stages. At E16, radial and non-radial processes were clearly associated with migrating neurons identified by the neuronal markers calretinin and gamma-aminobutyric acid. Non-radial glial fibers were no longer evident by E19. Radial fibers were gradually replaced by immature astrocytes at the end of embryonic development. In the perinatal period, vimentin immunoreactivity labeled immature astrocytes and then gradually decreased; vimentin-immunopositive cells were only found in the internal capsule by the second postnatal week. Glial fibrillary acidic protein immunoreactivity appeared at birth in astrocytes of the internal capsule, but was not evident in most of the adult thalamic nuclei. Confocal and immunoelectron microscopy allowed direct examination of the relationships between neurons and glial processes in the embryonic thalamus, showing the coupling of neuronal membranes with both radial and non-radial glia during migration. Peculiar ultrastructural features of radial glia processes were observed. The occurrence of non-radial migration was confirmed by carbocyanine-labeled neuroblasts in E15 cultured slices. The data provide evidence that migrating thalamic cells follow both radial and non-radial glial pathways toward their destination.     

Glial expression of estrogen and androgen receptors after rat brain injury

Estrogens and androgens can protect neurons from death caused by injury to the central nervous system. Astrocytes and microglia are major players in events triggered by neural lesions. To determine whether glia are direct targets of estrogens or androgens after neural insults, steroid receptor expression in glial cells was assessed in two different lesion models. An excitotoxic injury to the hippocampus or a stab wound to the parietal cortex and hippocampus was performed in male rats, and the resultant expression of steroid receptors in glial cells was assessed using double-label immunohistochemistry. Both lesions induced the expression of estrogen receptors (ERs) and androgen receptors (ARs) in glial cells. ERalpha was expressed in astrocytes immunoreactive (ERalpha-ir) for glial fibrillary acidic protein or vimentin. AR immunoreactivity colocalized with microglial markers, such as Griffonia simplicifolia lectin-1 or OX-6. The time course of ER and AR expression in glia was studied in the stab wound model. ERalpha-ir astrocytes and AR-ir microglia were observed 3 days after lesion. The number of ERalpha-ir and AR-ir glial cells reached a maximum 7 days after lesion and returned to low levels by 28 days postinjury. The studies of ERbeta expression in glia were inconclusive; different results were obtained with different antibodies. In sum, these results suggest that reactive astrocytes and reactive microglia are a direct target for estrogens and androgens, respectively.     

Vestibular compensation after ganglionectomy: ultrastructural study of the tangential vestibular nucleus and behavioral study of the hatchling chick.

The tangential nucleus is a major part of the avian vestibular nuclear complex, and its principal cells are structurally distinctive neurons participating in the vestibuloocular and vestibulocollic reflexes. After unilateral peripheral vestibular lesion, a behavioral recovery of function defined as vestibular compensation is observed. Because sprouting and hypertrophy of synapses have been reported in other regions of immature animals after central nervous system injury, we investigated whether this also occurs in the vestibular nuclei during compensation. To test this hypothesis, unilateral vestibular ganglionectomy was performed on 4-6-day-old hatchlings and vestibular function was tested during the next 2 months. Degeneration and evidence for regeneration of synapses were studied in the tangential nucleus at 1, 3, 7, and 56 days after surgery. Spoon endings, large vestibular terminals on the principal somata, degenerated 1-3 days after surgery. However, the small synaptic terminals showed no significant change in the percentage or number covering the soma or in mean terminal lengths in the deafferented or contralateral tangential nucleus. Furthermore, there was no evidence of neuron death in the tangential nucleus. Vestibular compensation occurred in three stages: 0-3 days, when vestibular synapses degenerated and severe behavioral deficits were seen; 4-9 days, when primary vestibular fibers degenerated centrally and marked improvement in both the static and the dynamic symptoms were observed; and 10-56 days, when changes in neuronal morphology were not detected but the dynamic symptoms gradually improved. Accordingly, after unilateral vestibular ganglionectomy, vestibular compensation proceeded without ultrastructural evidence of sprouting or hypertrophy of axosomatic synapses in the hatchling tangential nucleus. This rapid behavioral recovery of function distinguishes the vestibular system from other sensory systems, which, in general, exhibit much less robust recovery after injury to their peripheral receptors.     

Changes in brainstem calcitonin gene-related peptide after VIIth and VIIIth cranial nerve lesions in guinea pig

The present study investigated the effect of seventh and eighth cranial nerve lesions on the prominence of calcitonin gene-related peptide in the hypoglossal (XII), facial (VII), abducens (VI), and oculomotor (III) cranial nerve nuclei. Guinea pigs were anesthetized and subjected to unilateral cochlear removal, vestibular end organ ablation, and seventh nerve transection. After a survival period ranging from 4 h to 5 days, each animal was anesthetized and perfused intracardially. Frozen sections were collected through the brainstem and stained immunohistochemically for calcitonin gene-related peptide using a polyclonal antibody with the Vectastain ABC kit and protocol. Positive cells were counted in each nucleus bilaterally and analyzed for side to side differences.Nuclei XII and III showed no significant difference in the numbers of cells staining positively for calcitonin gene-related peptide between the ipsilateral and the contralateral sides to the lesion. However, nuclei VII and VI showed elevated numbers ipsilateral to the lesion on some days, but not all. For VII, there was no significant difference before 24 h, but there were significant differences 1–5 days after the lesion. Similarly, in VI, there was no difference before 24 h, but differences were significant beginning with day 1 and continuing through day 3, and finally disappearing by day 4.Changes in the numbers of CGRP positive cells in VII measurable 24 h after the lesion and continuing for at least 5 days afterward indicate a central nervous system retrograde response to peripheral motor nerve injury. However, since no peripheral damage occurred to any structure other than those related to VII and VIII, increased numbers of calcitonin gene-related peptide positive cells in VI indicates the presence of a separate mediating mechanism. We believe this increase may be due, not to the direct loss of a peripheral nerve as in the case of VII, but instead, to an indirect motor stimulation of the eye muscles (indicated by nystagmus) that accompanies unilateral vestibular damage. Thus, while the central CGRP response in VII is activated by a retrograde neuronal mechanism, the central calcitonin gene-related peptide response in VI is activated by an anterorade transsynaptic neuronal mechanism.     

Immunohistochemical localization of phospholipase D1 in rat central nervous system

Phospholipase D (PLD) is one of the intracellular signal transduction enzymes and plays an important role in a variety of cellular functions. We investigated the distribution of PLD isozyme, PLD1 in the rat brain and spinal cord using an immunological approach. Western blot analysis showed the presence of PLD1 protein in all tissues studied, with significantly higher levels in the brainstem and spinal cord, which was correlated with the results obtained from PLD activity assay. Prominent and specific signals of PLD1 were observed in many functionally diverse brain areas, including the olfactory bulb, medial septum-diagonal band complex, cerebral cortex, brainstem, cerebellum, and spinal cord. In the brainstem, the red nucleus, substantia nigra, interpeduncular nucleus, cranial motor nuclei (trigeminal motor, abducent, facial, and hypoglossal), sensory cranial nerve nuclei (spinal trigeminal, vestibular, and cochlear), as well as nuclei of the reticular formation, all showed intense immunoreactivity. Purkinje cells and deep cerebellar nuclei of the cerebellum were also labeled intensely. However, no significant labeling was found in the thalamus, epithalamus, and basal ganglia. Although many of the PLD1 immunoreactive cells were neurons, PLD1 was also expressed in glial cells such as presumed astrocytes and tanycytes. These findings suggest that PLD1 may play an important role in the central nervous system of the adult rat.     

Protein SP40,40-like Immunoreactivity in the Rat Brain: Progressive Increase With Age

The pattern of distribution of SP40,40-like immunoreactive structures has been studied in the rat brain using a well-characterized polyclonal antibody raised against the SP40,40 protein. Protein SP40,40 is the human counterpart of the rat sulphated glycoprotein 2, whose mRNA shows widespread expression in the developing and mature brain. In young adult rats few immunoreactive structures were observed. Some immunoreactive neurons were found in the cingulate cortex, the arcuate and perifornical hypothalamic nuclei, as well as glial labelling in the hypothalamus. A striking increase in the number of immunoreactive cells was observed as a function of age. In 20–22-month-old rats, numerous immunoreactive cells were observed in the cingulate cortex, several thalamic and hypothalamic nuclei, the red nucleus, olivary nuclei, superior colliculus, and many cranial nerve nuclei. Whereas the immunoreactivity was restricted to a diffuse labelling of the cell bodies and processes in young rats, other forms of labelling were observed in aged rats: punctate cytoplasmic labelling and intensely stained granules with no visible cell membrane. A further increase in the density of the immunoreactive material was observed in 30–31-month-old rats. Double labelling experiments demonstrated that the SP40,40 immunoreactivity was almost exclusively located in neurons and not in glial cells (with the exception noted above). The distribution of SP40,40 immunoreactivity in aged rats did not coincide with the distribution of the microtubule-associated tau protein, OX42 or lipofuscin.     

Temporary sensory deprivation changes calcium-binding proteins levels in the auditory brainstem

Auditory brainstem neurons probably depend on afferent input to maintain calcium homeostasis within a narrow range. These neurons are endowed with high concentrations of the calcium-binding proteins parvalbumin, calretinin, and calbindin D28k that are presumed to buffer cytosolic calcium transients. To determine the effects of functional deafferentation on these proteins in the auditory brainstem of adult guinea pigs, we have manipulated the sensory input with an intracochlear perfusion of the glutamate agonist α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), which is known to transiently disconnect inner hair cells and primary auditory dendrites. Semiquantitative measures of immunostaining intensities showed rapid and reversible changes in calcium-binding protein levels. By 24 hours after AMPA treatment, calretinin immunostaining was reduced in deafferented neurons of the cochlear nuclei and their axons in the superior olivary nuclei. In contrast, calbindin D28k immunoreactivity levels by this time were higher in deafferented neurons of the medial nucleus of the trapezoid body and their axons in the lateral superior olivary nucleus (LSO). Parvalbumin immunostaining was also generally increased in deafferented neurons, but changes were less evident and more complex. The changes in all three immunoreactivities disappeared with the progressive restoration of afferent input. Normal levels were reestablished by 5 days after AMPA treatment, when afferent activity had almost completely recovered. These results show that calcium-binding protein immunostaining in auditory neurons is functionally responsive to afferent activity. The increased buffering capacity in deafferented neurons as shown by the rises in parvalbumin and calbindin D28k immunostaining may be part of mechanisms promoting neuronal survival after loss of sensory input. This input, on the other hand, may be necessary for maintaining the high calretinin levels normally present in cochlear nucleus neurons. J. Comp. Neurol. 378:1–15, 1997. © 1997 Wiley-Liss, Inc.     

Immunohistochemical Localization of Kininogen in Rat Spinal Cord and Brain

Kininogen localization has been determined by immunocytochemistry in rat spinal cord and brain using a kinin-directed kininogen monoclonal antibody. In the spinal cord, there were immunostained neurons and fibers in laminae I, II, VII, and IX, intensely stained fibers in the superficial layers of the dorsal horn, and immunoreactive glial and endothelial cells. Small neurons, satellite cells, and Schwann cells immunostained distinctly in the dorsal root ganglion. In the brain stem, there were immunoreactive neurons and fibers in the tractus solitarius and nucleus, trigeminal spinal tract and nuclei, periaqueductal gray matter, vestibular nuclei, cochlear nuclei, trapezoid body, medial geniculate nucleus, and red nucleus. Immunostained neurons and fibers were also found in cerebellum (dentate nucleus), cerebral cortex (layers III and V), hippocampus (pyramidal cell layer), and corpus callosum. Glia and endothelial cells stained in all brain regions. The widespread location of kininogen in neurons and their processes, as well as in glial and endothelial cells, indicates more than one functional role, including those proposed as a mediator, a calpain inhibitor, and a kinin precursor, in a variety of neural activities and responses.     

Regulation of NMDA Receptor Subunit mRNA Expression in the Guinea Pig Vestibular Nuclei Following Unilateral Labyrinthectomy

The localization of neurons expressing mRNAs for the NRI and NR2A-D subunits of the glutamatergic NMDA receptor was examined by non-radioactive in situ hybridization throughout the guinea pig vestibular nuclei. After deafferentation of the vestibular nuclei by unilateral labyrinthectomy, modifications of the mRNA distributions were followed for 30 days. A quantitative analysis was performed in the medial vestibular nucleus by comparison of the labelled neurons in the ipsi- and contra-lateral nuclei. In vestibular nuclei, the NR1 subunit mRNA was found in various populations of neurons. The NR2A and NR2C subunit mRNAs were less widely distributed, whereas little NR2D mRNA was detected and only rare cells contained NR2B mRNA. NRI and NR2A-D mRNAs were colocalized in some but not other neuronal types. Twenty hours after the lesion, there was a transient ipsilateral increase of NR1 mRNA level in the medial vestibular nucleus, followed by a decrease 48 h after the lesion and, at 3 days, by recovery to the control level. An ipsilateral increase in the mRNA level of NR2C subunit was detected 20 h after lesion and maintained at 48 h. No significant changes were apparent in NR2A, NR2B and NR2D mRNA levels. The distributions and the differential signal intensities of NR2A-D mRNAs suggest various subunit organizations of the NMDA receptors in different neurons of the vestibular nuclei. Neuronal plasticity reorganizations in the vestibular nuclei following unilateral labyrinthectomy appear to include only changes in NR1 and NR2C mRNA levels modifying the functional diversity of the NMDA receptor in the ipsilateral medial vestibular nucleus neurons. The transient changes in NRI and the NR2C subunit mRNA expressions in response to sensory deprivation are consistent with an active role for NMDA receptors in the appearance and development of the vestibular compensatory process.