Immunocytochemical localization of the insulin-responsive glucose transporter 4 (Glut4) in the rat central nervous system.

We have previously reported that the insulin-responsive glucose transporter GLUT4 is strongly expressed by discrete areas of the rat brain (Leloup et al. [1996] Molec. Brain Res. 38:45-53). In the present study, a sensitive immunocytochemical technique has been used to analyze extensively the anatomical and ultrastructural localizations of GLUT4 in the rat central nervous system in order to gain insight into the physiological role of this transporter. We confirm that GLUT4 is expressed by numerous neurons of the brain and spinal cord, whereas glial cells are more scarcely labeled. In both light and electron microscopy, we observe that the immunoreactivity for GLUT4 is localized mainly in the somatodendritic portion of neurons, where some cisterns of rough endoplasmic reticulum, ribosomal rosettes, certain Golgi saccules, and some intracytoplasmic vesicles are labeled. In contrast, axons and nerve terminals are only occasionally immunostained in certain brain regions such as the neocortex and the ventricular surfaces for example. The GLUT4-immunoreactive structures appear concentrated and most prominently immunostained in motor areas, such as the sensorimotor cortex, most basal ganglia and related nuclei, the cerebellum and deep cerebellar nuclei, a number of reticular fields, motor nuclei of cranial nerves, and motor neurons of the ventral horn of the spinal cord. The labeled regions, which also include some sensory nuclei, are often those in which Vissing et al. ([1996] J. Cerebral Blood Flow Metab. 16:729-736) have shown that exercise stimulates local cerebral glucose utilization, so that GLUT4 might be involved in this effect. On the other hand, the fact that the anatomical localizations of GLUT4 reported here generally agree with the distribution of insulin- or insulin-receptor- related receptors is important since it indicates that the translocation of GLUT4 might also be regulated by insulin in the central nervous system.

Opposite changes in the expression of enkephalin in the amygdala and hypothalamus after lesions of the bed nucleus of the stria terminalis in the rat.

The expression of enkephalin in neurons of the rat forebrain was studied by in situ hybridization and immunohistochemistry after unilateral injections of ibotenic acid into the bed nucleus of the stria terminalis. Initially, we observed that the destruction of nerve cell bodies in this nucleus resulted in a prominent bilateral increase in the number of neuronal perikarya immunoreactive for [Met]enkephalin in the lateral/basolateral amygdaloid complex-especially in the anterior division of the latter nucleus-as compared with NaCl-injected rats. In a separate set of experiments, this effect was associated with a significant (two times) enhancement of the number of nerve cell bodies containing preproenkephalin A messenger RNAs in the same amygdaloid nucleus ipsilateral to the injection, as compared with controls. In the hypothalamus of both experimental and control rats, the nerve cell bodies immunoreactive for [Met]enkephalin were few since the animals were not pretreated with colchicine, and the effects of the lesion were difficult to appreciate. However, using in situ hybridization, numerous nerve cell bodies containing preproenkephalin A messenger RNAs were detected bilaterally in the perifornical area, the paraventricular (parvocellular division) and the ventromedial nuclei of the hypothalamus. In the latter nucleus, the lesion of the bed nucleus of the stria terminalis resulted in a strong decrease (about two times) in the number of labelled cell bodies as compared with the controls, whereas no significant changes were found bilaterally in the paraventricular nucleus. In agreement with some data of the literature, our results indicate that the bed nucleus of the stria terminalis plays an important role in the regulation of neuropeptide genes expression in certain regions of the limbic system. Such a role is often exerted by nerve fibres afferents to the nerve cell bodies considered. However, from numerous neuroanatomical data of the literature, it appears more probable that the induction or inhibition of the expression of enkephalin in presynaptic neurons is due to the disappearance of their postsynaptic target in the bed nucleus of the stria terminalis.

Discrete brain areas express the insulin-responsive glucose transporter GLUT4.

Whether or not glucose utilization in the brain is insulin-dependent is still a controversial issue. We looked for the presence of the insulin-sensitive glucose transporter (GLUT4) in rat brain and obtained the following results: (1) poly(A) RNAs from the hypothalamus and anterior medulla oblongata hybridize with a cDNA probe for GLUT4; (2) reverse transcription-polymerase chain reaction (RT-PCR) on RNA from various brain nuclei detects GLUT4 transcripts; (3) immunocytochemistry, using a polyclonal antibody to GLUT4; reveals a specific immunostaining pattern, whereas both electronic microscopy and double immunofluorescence staining, using a neurofilament protein marker, indicate a neuronal localization. These results are discussed in terms of a putative neuromodulator role of insulin, via glucose utilization, in brain areas involved in the regulation of fuel metabolism.

Glucose transporter 2 (GLUT 2): expression in specific brain nuclei.

In the brain, certain neurons appear to be sensitive to changes in local and/or plasma glucose concentration. The alterations in the electrical activity of these neurons probably depend on the existence of 'glucose sensors', which may be one of the glucose transporters described so far. Because of suitable kinetic properties, we hypothesized that the glucose transporter 2 (GLUT 2) may well constitute one of the cerebral 'glucose sensors'. In this study, it was demonstrated, using the polymerase chain reaction, that GLUT 2 mRNAs are present in a limited number of brain nuclei, including the nucleus tractus solitarius, the motor nucleus of the vagus, the paraventricular hypothalamic nucleus, the lateral hypothalamic area, the arcuate nucleus and the olfactory bulbs. These localizations were confirmed by immunocytochemistry, but the cerebral distribution of GLUT 2-like immunoreactivity was far larger than initially expected. Furthermore, electron microscopic observations showed that, within the regions examined, GLUT 2 was localized to a restricted population of astrocytes. The localization of GLUT 2 in regions previously connected with feeding behavior supports an indirect role for GLUT 2 in 'glucose sensing' in these specific cerebral structures.

Demonstration of peptidergic afferents to the bed nucleus of the stria terminalis using local injections of colchicine. A combined immunohistochemical and retrograde tracing study.

In the present study, we demonstrate the existence of numerous peptidergic afferents to the bed nucleus of the stria terminalis (BNST) using the retrograde transport of gold-labeled wheat germ agglutinin-apo-peroxidase (G-WGA-HRP) combined with the indirect immunoperoxidase method after intraparenchymatous injections of colchicine. At first, we show that local injections of colchicine alone into the BNST are able to induce the retrograde accumulation of peptides until the nerve cell bodies of origin, probably because of the blockade of axonal transport in nerve terminal arborizations innervating this nucleus. The actual existence of putative peptidergic afferents to the BNST indicated by the local injections of colchicine was established using: a) the retrograde transport of G-WGA-HRP from the BNST combined with immunocytochemistry after administration of colchicine at the same place, b) the anterograde "transport" of the fluorescent tracer DiI from selected nuclei of the forebrain. We demonstrate that the neurons immunoreactive for enkephalins, neurotensin, or substance P that innervate the BNST are localized mainly in the central amygdaloid nucleus, the paraventricular thalamic nucleus, and the ventromedial hypothalamic nucleus ipsilateral to the injection, as well as bilaterally in the magnocellular paraventricular and perifornical regions of the hypothalamus. From these results it may be concluded that intracerebral injections of colchicine constitute a powerful tool to search for multiple peptidergic afferents to a given brain nucleus using only immunohistochemistry. The existence of these pathways, however, must be verified by other neuroanatomical methods because of the problem of nerve fibers of passage.

Effects of intranigral injections of colchicine on the expression of some neuropeptides in the rat forebrain: an immunohistochemical and in situ hybridization study.

In the present study, we describe the neurochemical effects of intranigral injections of colchicine in the rat forebrain using immunohistochemistry and in situ hybridization. The observations on the injected side are compared to the contralateral one and to the sham-operated rats. We demonstrate that such injections are able to strongly enhance the immunoreactivity for Met-enkephalin (ME), substance P (SP) and neuropeptide Y (NPY) in numerous nerve cell bodies of the limbic system (injected side), whereas the levels of the corresponding mRNAs are differently modified according to the region examined. A clear correlation between the enhancement of the immunostaining for ME and SP and that of the preproenkephalin (PPA) and preprotachychinin gene transcripts was observed in neuronal perikarya of the medial amygdaloid nucleus (SP), of the dorsolateral hypothalamus (ME) and of the ventromedial hypothalamic nucleus (SP). These observations are interpreted as an induction--or increased expression--of neuropeptide genes in neuronal perikarya postsynaptic to nerve fibers originating in the midbrain and brain stem. In this case, colchicine is thought to block the electrophysiological activity of ascending nerve fibers (anterograde and postsynaptic effect). In the case where the enhancement of the immunoreactivity for the studied neuropeptides was associated with no change or a decreased expression of the corresponding genes in the same brain areas, colchicine may have blocked the axoplasmic transport of peptides in nerve fibers projecting to the midbrain and/or brain stem (6). This may result in a retrograde accumulation of peptides in the nerve cell bodies of origin and, eventually, in a negative feedback regulation of the corresponding encoding genes in these perikarya (retrograde and presynaptic effect of colchicine). The drastic behavioral effects of bilateral intranigral injections of colchicine, on ingestive behavior in particular, have been studied in a following paper.

Forebrain connections of the rat paraventricular thalamic nucleus as demonstrated using the carbocyanide dye DiI.

The anatomical connections of the rat paraventricular thalamic nucleus (PaVT) have been studied using the fluorescent dye DiI. The fact that this compound is able to dissolve and to diffuse in plasma membranes in formalin-fixed tissues (Godement et al., 1987) allowed us to depose precisely tiny quantities of DiI on the PaVT, in order to study its neuronal connections. Ventralward, the forebrain projections of the PaVT were directed to the reuniens nucleus of the thalamus, the dorso-medial hypothalamic region, the zona incerta and the lateral hypothalamic area. In the rostral direction, the PaVT appeared to innervate mainly the medial preoptic area, the bed nucleus of the stria terminalis (particularly the ventral and medial regions) and the medial septum. The forebrain afferent connections of the PaVT were found to originate mainly in the lateral and periventricular hypothalamic area, the medial preoptic area, the bed nucleus of the stria terminalis and the medial septum where the fluorescent nerve cell bodies appeared particularly numerous. From these results, it is concluded that DiI is a useful tool for demonstrating the neuroanatomical connections of small brain nuclei, as well as for post-mortem neuropathological studies.

Afferent connections of the rat substantia nigra pars lateralis with special reference to peptide-containing neurons of the amygdalo-nigral pathway.

The afferent connections of the rat substantia nigra pars lateralis have been studied using the retrograde axonal transport of fluorescent latex microspheres. The most numerous groups of retrogradely labelled nerve cell bodies were observed bilaterally in the parabrachial complex and several hypothalamic nuclei, whereas the parietal neocortex, the fundus striati, the central nucleus of the amygdala and the bed nucleus of the stria terminalis were labelled on the injected side only. The neuronal projections from the central amygdaloid nucleus to the substantia nigra pars lateralis and lateral part of the rostral pars compacta have additionally been confirmed by anterograde tracing using wheat-germ agglutinin coupled to horseradish peroxidase. The presence of some peptides in this pathway was studied by combining the use of the same retrograde tracer with immunofluorescence after intra-amygdaloid injections of colchicine. With this method, we have demonstrated that Met-enkephalin, dynorphin and neurotensin are probably utilized as neurotransmitters or co-transmitters in the neurons of the amygdalo-nigral pathway.

Enkephalin-containing nerve cell bodies in the substantia nigra of the rat: demonstration by immunohistochemistry and in situ hybridization.

The use of a new and very sensitive immunohistochemical method, combined with intracerebral injections of colchicine, has allowed us to show that a number of nerve cell bodies immunoreactive for Met-enkephalin are present in several mesencephalic nuclei of the rat, including the different subdivisions of the substantia nigra (SN). The existence of numerous neuronal somata of this kind in the medial part of the SN pars compacta and in the lateral half of the pars reticulata is rather new. The latter has been ascertained by demonstrating a perikaryal immunoreactivity for synenkephalin in the same regions of the SN. In addition, by in situ hybridization, we have shown that neuronal cell bodies expressing the preproenkephalin A (PPA) gene are also present in the same regions of the SN. However, the fact that a strong radioautographic reaction was found only in rats which received an intranigral injection of 6-hydroxydopamine indicates that these neurons are probably not dopaminergic and that an induction of the PPA gene occurs in these animals.

Origin of some enkephalin-containing afferents to the ventro-medial region of the globus pallidus in the rat.

The retrograde transport of WGA-HRP adsorbed to colloidal gold was combined with the indirect immunoperoxidase technique to study the origin of enkephalin-containing afferents to the medial and ventral regions of the globus pallidus (GP). On the injected side, the nerve cell bodies labeled retrogradely or double labeled were numerous in the central nucleus of the amygdala (ACe), scattered in the bed nucleus stria terminalis (BNST) and few in the fundus striati. In the ACe, approximately 40% and 20% of the retrogradely labeled perikarya were found immunoreactive for Met-enkephalin and Leu-enkephalin, respectively, whereas they were only 30% and 15% in the BNST. It is concluded that the enkephalinergic afferents of the ventro-medial region of GP, which contains the basal nucleus of Meynert in the rat, are largely of limbic origin.

Modifications of precentral cortex discharge and EMG activity in monkeys with MPTP-induced lesions of DA nigral neurons.

1. Individual neurons were recorded extracellularly in the precentral forelimb area of two monkeys trained to perform rapid, large amplitude flexion and extension movements of the contralateral forearm in response to auditory signals. Electromyographic (EMG) activity in the biceps/triceps muscles was recorded separately under the same conditions. The dopaminergic (DA) neurons of the substantia nigra (SN) were destroyed selectively by repeated series of intravenous injections of MPTP. The lesion was verified on serial slices using both tyrosine hydroxylase immunocytochemistry and classical staining methods. 2. In normal monkeys, the frequency of firing of precentral neurons shows rapid changes shortly before the onset of displacement. In our sample (n = 102), most of the neurons (49%) tested during movement in both directions (flexion, extension) showed a reciprocal pattern of activity for the two directions of movement, a small percentage (19%) exhibited a change for only one direction (unidirectional neurons), and the remaining 32% displayed a similar change for both directions of movement (bidirectional neurons). 3. In MPTP-treated monkeys, movement-related modification of neuronal activity was more gradual, beginning earlier and lasting longer relative to the onset of movement. The cellular reaction time (the time between the auditory cue and a significant change in neuronal activity) was not significantly altered. Spontaneous firing of precentral neurons (n = 124) did not increase significantly, and the dynamic discharge rate was unchanged after the nigral lesion. However, only 18% of cortical neurons still presented a reciprocal pattern of discharge for the two directions of movement, while the percentage of unidirectional neurons increased (50%), and the percentage of bidirectional neurons remained the same (32%). 4. After MPTP treatment, alterations in movement parameters and EMG activity were observed. Mean reaction time and movement duration increased by 20-25% and 25-30% respectively. The movements were slower and were associated with a generalized depression in the shape and the amplitude of EMG activity in the agonist muscle. 5. The neuronal basis for the observed central and peripheral disturbance in the MPTP-treated monkeys is discussed. We conclude that SN lesion leads to two main disturbances of cortical activity: i) the loss of the reciprocal pattern of response of movement-related cortical cells, and ii) an inability of the motor cortex to modify its activity in response to peripheral input.

Increase in paradoxical sleep after destruction of serotoninergic innervation in the nucleus tractus solitarius of the rat.

The evolution of paradoxical sleep, slow-wave sleep and arterial pressure was studied following microinjection of 5,7-dihydroxytryptamine in the nucleus tractus solitarius in rats. The extent of the lesions was assessed using immunohistochemistry for serotonin. Global lesions of serotoninergic nerve terminals of the intermediate and commissural regions of the nucleus produced an important and long-lasting increase in paradoxical sleep (+50-70%), a decrease in slow-wave sleep (-20%) and a moderate increase of arterial pressure during all states of the sleep-wake cycle. In addition, more discrete lesions indicated that only the lesion of the area near the obex produced the longer term increase of paradoxical sleep whereas only the lesion of the commissural region of the nucleus produced the long-term decrease of slow-wave sleep. These data demonstrate that serotoninergic projections to the nucleus tractus solitarius exert a regulatory influence upon the specific mechanisms responsible for paradoxical sleep and slow-wave sleep in rats. Furthermore, they suggest that serotonin within the nucleus tractus solitarius plays an important role in the homeostatic cardiovascular and sleep-wake-cycle regulation in rats.

Immunohistochemical study of catecholaminergic cell bodies in the rat spinal cord.

Immunohistochemistry of three specific synthesizing catecholamine enzymes was used in the rat spinal cord to determine precisely the distribution of catecholaminergic perikarya and the nature of the neurotransmitter they contain. Single and double labeling experiments were performed on cryostat sections from perfused rats. The peroxidase anti-peroxidase (PAP) and the indirect fluorescence techniques were used for labeling spinal catecholaminergic somata and separated into two completely different populations. The first is located in the upper cervical cord and includes three apparently distinct groups: a lateral cluster, of probably a noradrenergic nature, and two central subgroups where noradrenergic and dopaminergic neurons are intermingled. It is likely that these cervical cells represent caudal extensions of the medullary catecholaminergic cell groups. In the remaining cord, only tyrosine hydroxylase immunoreactive cell bodies have been found. Accordingly, this second population is probably dopaminergic. It is present almost exclusively in the first sacral segments, where it is located in the commissural (mostly lateral) grey matter and in the marginal dorsal horn.

[Immunocytochemical and autoradiographic research on the growth of serotoninergic fibers to the cerebral ventricles in the perinatal period in rats]. / Immunotsitokhimicheskoe i avtoradiograficheskoe issledovaniia prorastaniia serotoninergicheskikh volokon k zheludochkam mozga v perinatal'nom periode u krys.

Anatomical relationships between serotoninergic (5-HT) fibers and cerebral ventricles were studied in rats from the 16th fetal day until the 9th postnatal day with immunocytochemistry and radioautography. In the latter case, 5-HT neuronal elements were detected according to their specific uptake of intraventricularly injected 3H-5-HT. On the 16th fetal day, occasional 5-HT fibers first spread from the main place of their origin in the raphe nuclei to the dorsocaudal portion of the 3rd ventricle and aqueduct. Two days later, a more extensive network of 5-HT fibers appeared around the dorsal portion of the 3rd ventricle, whereas fibers only rarely penetrated fibers became noticeable in the lateral and 3rd ventricles. The functional significance of hypothalamic and ventricular 5-HT is discussed from the standpoint of its being either a modulator of growth and differentiation of the developing brain, or a factor involved in some specific neuroendocrine functions.

Immunocytochemical and radioautographic study of serotonin projections to cerebral ventricles of perinatal rats.

Anatomical relationships between serotoninergic (5-HT) fibers and cerebral ventricles were studied in rats from the 16th fetal day until the 9th postnatal day with immunocytochemistry and radioautography. In the last case, 5-HT neuronal elements were detected according to their specific uptake of intraventricularly injected [3H]5-HT. At the 16th fetal day, occasional 5-HT fibers first spread from the main place of their origin in the raphe nuclei to the dorsocaudal portion of the 3rd ventricle and aqueduct. Two days later, a more extensive network of 5-HT fibers appeared around the dorsal portion of the 3rd ventricle whereas fibers only rarely penetrated toward its ventral portion. By the 9th postnatal day, extensive networks of supraependymal fibers became noticeable in the lateral ventricles and in the dorsal portion of the 3rd ventricle. In addition, a number of 5-HT fibers surrounded the infundibular and preoptic recesses and sometimes penetrated to the ventricular cavity. The functional significance of hypothalamic and ventricular 5-HT as a modulator of either the growth and differentiation of the developing brain or of some specific neuroendocrine functions is discussed.

Immunohistochemical demonstration of catecholaminergic cell bodies in the spinal cord of the rat. Preliminary note.

In order to elucidate the anatomy of the spinal dopaminergic system, an immunohistochemical study using a tyrosine-hydroxylase (TH) antibody was undertaken in the rat. Intracisternal 6-hydroxydopamine (6-OHDA) injections were administered to destroy most of the noradrenergic fibres that descend to the spinal cord while preserving the dopaminergic fibres. The density of the remaining TH-like immunoreactive fibres was relatively low at all levels of the spinal cord; the highest density was observed in layers III, IV and X. In addition, we report the first evidence for the existence of TH-like immunoreactive cell bodies at definite levels (especially sacral) of the spinal cord.

Ultrastructural morphology of dopaminergic nerve terminals and synapses in the striatum of the rat using tyrosine hydroxylase immunocytochemistry: a topographical study.

Structures immunoreactive for TH were examined in the rat striatum (including caudate-putamen, nucleus accumbens and globus pallidus) by electron microscopy using the indirect peroxidase-labeled antibody method. Axon profiles and nerve terminals were the only structures stained by DAB precipitates in the axoplasm. The reactive boutons frequently contained a population of large pleomorphic vesicles (40-60 nm in diameter) but their interiors remained free of reactions. The synaptic contacts formed belonged principally to the symmetric type 2 of Gray while asymmetric Gray's type 1 synapses were rarely observed. The former were mostly apposed to dendritic trunks (rarely to perikarya) and the latter to dendritic spines. In addition, numerous immunoreactive nerve terminals were often found in close contact with small structures identified as the neck of dendritic spines. The active zone of these presumed synapses was characterized by a prominent thickening of the presynaptic membrane but the post-synaptic thickening was lacking. For similar reasons, it was difficult to assert the existence of one axo-axonic synapse when a positive nerve terminal was closely apposed to another one (generally unreactive). The exact morphology of dopaminergic synapses, or even their existence, have not been firmly established owing to large discrepancies between previous reports. No synapses, or synaptic contacts of either asymmetric or symmetric type were described according to the technique used. Our work was undertaken to elucidate further this problem, and in particular, we thought that regional differences in the synaptic organization might explain the divergent data. However, regional quantitative analysis performed in this study did not show significant differences in the percentage of either kind of synapses in the various striatal regions.

Ultrastructural study of cholecystokinin-like immunoreactive nerve terminals in the rat nucleus accumbens.

The cholecystokinin (CCK)-like immunoreactive nerve terminals were studied in the caudal and medial parts of the rat nucleus accumbens (NA), using the indirect immunoperoxidase technique, at the electron microscopic level. In the labelled axon terminals the immunoprecipitate is localized inside large dense-cored vesicles which are occasionally present, and surrounds small and medium-sized, round, clear synaptic vesicles. The immunoreactive nerve terminals participate in synapses of both asymmetrical and symmetrical types containing mostly small synaptic vesicles. The asymmetrical synapses are much more numerous and mainly axo-spinous. The symmetrical synapses are less frequent and are axo-dendritic or axo-somatic.

Different localizations of Met-enkephalin-like immunoreactivity in rat forebrain and spinal cord using hydrogen peroxide and Triton X-100. Light microscopic study.

The histological distribution of met-enkephalin-like immunoreactivity was studied in the forebrain (particularly the striatum) and the spinal cord of the rat using the indirect peroxidase-labelled antibody method. In most experiments, vibratome sections of formaldehyde-fixed tissues and purified antibodies were used. The search for optimal conditions for the immunohistochemical reaction lead us to establish that met-enkephalin-containing perikarya of both untreated and colchicinized animals were better demonstrated when tissue were pre-treated with diluted hydrogen peroxide only. The additional treatment of these sections with Triton X-100 (or some other detergents) resulted in the near disappearance of the perikaryal immunoreactivity; on the contrary, numerous met-enkephalin containing nerve fibres and varicosities were then demonstrated in the same region. Using only the hydrogen peroxide treatment, we found numerous met-enkephalin-containing perikarya in the medial and ventral regions of the neostriatum. This distribution was prolonged caudally by the existence of a prominent group of stained somata in the ventral putamen-central nucleus of the amygdala. When intraventricular injections of colchicine were used, positive perikarya were more numerous within the striatum (the globus pallidus excepted) but their distribution was largely the same as in non injected animals. However, some new groups of somata were stained in this case in the forebrain (in the lateral septum, the olfactory tubercle and the hypothalamus particularly). In control animals only few met-enkephalin-containing perikarya were observed in the dorsal horn of the spinal cord when H2O2 pretreatment was used alone and they were numerous only when intraspinal injections of colchicine were performed. Met-enkephalin-containing fibres and varicosities, which were scattered in the whole neostriatum in the conditions used above, became very numerous when the tissue sections were incubated in the presence of Triton X-100. Their density increased markedly from the latero-dorsal to the medio-ventral regions but, in addition, an organization under the form of islands of stronger immunoreactivity was also evidenced. These islands were more numerous ventrally in the anterior neostriatum and in the central region of the "putamen." The dense plexus of immunoreactive nerve fibers forming "tube-like structures" which was always observed in the paleostriatum and in the cranial medial forebrain bundle (islands of Calleja) appeared more diffuse when detergents were used.(ABSTRACT TRUNCATED AT 400 WORDS)