Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats

Five N-methyl-D-aspartate (NMDA) receptor subunits have been identified thus far: NR1, NR2A, NR2B, NR2C, and NR2D. Here, we have analyzed the expression patterns of mRNAs for the NMDA receptor subunits in the developing and adult rats by in situ hybridization. The developmental changes of the expression patterns were most salient in the cerebellum. In the external granular layer, hybridization signals of mRNAs, for NR1, NR2A, NR2B, and NR2C appeared by postnatal day 3, but no NR2D mRNA was expressed at any developmental stage examined. The NR1 mRNA was expressed in all cerebellar neurons at all developmental stages examined. The signals for the NR2A mRNA appeared in Purkinje cells and granule cells during the second postnatal week. The signals for the NR2B mRNA in granule cells were seen transiently during the first 2 weeks after birth. The signals for NR2C mRNA appeared in granule cells and glial cells during the second postnatal week. The signals for NR2D mRNA appeared transiently in Purkinje cells during the first 8 postnatal days; in adult rats, these were seen in stellate and Golgi cells. In the cerebellar nuclei, mRNAs for NR1, NR2A, NR2B, and NR2D were more or less expressed on postnatal day 0, while expression signals for the NR2C mRNA were first detected in postnatal day 14. Thus, the most conspicuous changes of expression patterns were observed in the cerebellar cortex during the first 2 weeks after birth, when development and maturation of the cerebellum proceed most rapidly. © 1994 Wiley-Liss, Inc.     

Distinct distribution and time-course changes in neuronal nitric oxide synthase and inducible NOS in the paraventricular nucleus following lipopolysaccharide injection

Nitric oxide (NO) is known to be involved in the modulation of neuroendocrine function. To clarify the role of different isoforms of NO synthase (NOS) in the neuroendocrine response to immune challenge, the expressions of neuronal NOS (nNOS) and inducible NOS (iNOS) genes in the hypothalamus following lipopolysaccharide (LPS) injection were examined using in situ hybridization. NOS activity was also determined by NADPH-diaphorase (NADPH-d) histochemistry. LPS (25 mg/kg) or sterile saline was injected intraperitoneally to male Wistar rats and the rats sacrificed 30 min, or 1, 2, 3, 5, 12 or 24 h after injection. nNOS mRNA expression in the paraventricular nucleus (PVN) was significantly increased 2 h after LPS injection. iNOS mRNA, which was not detected until 2 h after LPS injection, was significantly increased in the PVN 3 h after LPS injection. Both RNA expressions had returned to basal levels by 12 h after LPS injection. The number of NADPH-d positive cells was significantly increased 5 h after LPS injection. iNOS expression was more robust in parvocellular PVN, while nNOS was distributed mainly in the magnocellular PVN. Double in situ hybridization histochemistry revealed that some of the iNOS- (48.4%) or nNOS-positive cells (34. 3%) in the parvocellular PVN expressed CRF mRNA. The results demonstrate that LPS-induced sepsis causes significant increases in nNOS and iNOS gene expression with different time-courses and distributions, and that iNOS mRNA was more frequently co-localized with CRF-producing parvocellular neurons in the PVN. Thus, NO produced by iNOS and nNOS may play an important role in the neuroendocrine response to an immune challenge. Distinct differences in the distribution and time-course changes of iNOS and nNOS suggest different roles for the hypothalamic-pituitary-adrenal axis and/or neurohypophyseal system.     

Differential expression of GABAA/benzodiazepine receptor beta 1, beta 2, and beta 3 subunit mRNAs in the developing mouse cerebellum.

Gamma aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian cerebellum. Cerebellar granule, Purkinje, and deep nuclear neurons are known to receive GABAergic afferents. Since GABA exerts its inhibitory effects via GABA receptors, it is of interest to determine the temporal relationship between the formation of GABAergic synapses and the expression of genes coding for the GABA receptor. In a previous study, we have examined the developmental expression of binding sites for [3H]muscimol, which binds with high affinity to the beta subunits of the GABAA/benzodiazepine (GABAA/BZ) receptor. In the present study, [35S]cRNA probes were used to examine the appearance and distribution of GABAA/BZ beta 1, beta 2, and beta 3 subunit mRNAs in the developing C57BL/6 mouse cerebellum by in situ hybridization. In the adult cerebellum, the distribution of the three subunit mRNAs was clearly different, despite considerable overlap, and their temporal expression differed throughout postnatal development. The beta 1 hybridization signal appeared within the cerebellar cortex during the second postnatal week as a discrete band at the interface of the molecular and granule cell layers. Grains were distributed diffusely over small densely staining cells surrounding the Purkinje cells; relatively few grains were visible over Purkinje cell bodies themselves. This distribution may reflect an association with Bergmann glia or basket cells. The beta 2 and beta 3 hybridization signals were present considerably earlier than that of the beta 1 mRNA. The beta 2 signal was present at birth in the molecular/Purkinje cell layer; as development progressed, the signal became increasingly intense over both granule and Purkinje cells. At birth, the beta 3 subunit mRNA was present in the external germinal and molecular layers, later becoming largely localized within the granule cell layer. Dense beta 2 and beta 3 cRNA probe labeling was present over the adult granule cell layer. Moderate levels of beta 2 signal were seen over Purkinje cell bodies; considerably less labeling was observed with the beta 3 probe. The adult distribution of beta 2 and beta 3 cRNA probes showed good spatial correspondence with the known GABAA receptor beta subunit markers, [3H]-muscimol and the mAb 62-3G1 antibody, each being present within the granule cell layer. Our results indicate that the temporal expression of GABAA/BZ receptor beta subunit messages within a given cell type may be independently regulated, and that acquisition of the beta 2 and beta 3 mRNAs occurs before these cells become integrated into mature synaptic circuits.     

Role of Dopaminergic D2 Receptors in the Regulation of Glutamic Acid Decarboxylase Messenger RNA in the Striatum of the Rat

Levels of messenger RNA (mRNA) encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot and in situ hybridization analyses in the striatum of the rat, after chronic injections of two neuroleptics, sulpiride and haloperidol. The Northern blot analysis showed that the chronic injection of sulpiride at high doses (80 mg/kg, twice a day, 14 days) increased striatal GAD and PPE mRNA levels by 120% and 78% respectively, when compared to vehicle-injected rats. Haloperidol injections at relatively low doses (1 mg/kg, once a day, 14 days) produced parallel increases in GAD (40%) and PPE (52%) mRNA levels. After in situ hybridization densitometric measurements were performed on autoradiograms from rats treated with sulpiride, haloperidol or vehicle. The distribution of GAD and PPE mRNA signals in control rats was homogeneous along the rostrocaudal extension of the striatum. A similar increase was found along this axis after sulpiride (20%) and haloperidol (30%) treatments. The cellular observation of hybridization signals showed that grain density for GAD mRNA was increased in a majority of striatal cells after both treatments. By contrast, the PPE mRNA hybridization signal only increased in a subpopulation of neurons. The effects of such treatments were also analysed by measuring GAD activity in the striatum and in its output structures, the globus pallidus and the substantia nigra. After the administration of sulpiride, GAD activity was not modified in the striatum but increased in the globus pallidus (by 17%). After haloperidol treatment, GAD activity was increased in the globus pallidus (20%) and the substantia nigra (17%). It is concluded that the interruption of dopaminergic transmission, more precisely the D2 receptor blockade, promotes in striatopallidal neurons an increase in GAD mRNA accompanied by an increase in GAD activity and PPE mRNA. A possible regulation of GAD mRNA and GAD activity in striatonigral neurons is also discussed.     

Quantitative in situ hybridization analysis of glutamic acid decarboxylase messenger RNA in developing rat cerebellum.

The appearance and relative amounts of GAD mRNA in rat cerebellar neurons during postnatal development was studied by in situ hybridization. GAD mRNA content within all GABAergic neurons increased during the first month of postnatal development, but the degree and time course of the increase varied among different neuronal types. In newborn rats, GAD mRNA was present only in the prenatally-formed Purkinje and Golgi cells. GAD mRNA in Golgi cells had reached adult levels by postnatal day 14, while GAD mRNA levels in Purkinje cells reached adult levels one week later. Most basket cells expressed GAD mRNA by postnatal day 14, and final levels were attained one week later. Stellate cells in the bottom two-thirds of the molecular layer attained their final GAD mRNA content by postnatal day 21 whereas stellate cells in close proximity to the pial surface were not yet mature at this age. No GAD mRNA was detected within the external granular layer at any time during development. In adult rat, approximately 40% of cerebellar GAD mRNA was contained within the Purkinje cell population, 38% within the stellate cells, 17% within the basket cells, and only 5% within the Golgi cells. Increases in GAD mRNA within GABAergic neurons during cerebellar development correlated with the timing of neuronal maturation and synaptogenesis in these cell populations, suggesting that synaptic activity affects GAD gene expression in developing cerebellum.     

Increased expression of glutamic acid decarboxylase mRNA in rat substantia nigra after an ibotenic acid lesion in the caudate-putamen

In situ hybridization histochemistry and RNA blots were used to study expression of glutamic acid decarboxylase (GAD) mRNA in rat caudate-nucleus and substantia nigra. In situ hybridization combined with computerized image analysis revealed that in the intact substantia nigra reticulata the cross-section area of GAD mRNA positive neurons were 25% larger in the dorsolateral part as compared with the ventromedial part. A unilateral ibotenic acid injection in caudate-putamen lesioned neurons, some of which project to the ipsilateral substantia nigra. An increased level of GAD mRNA was observed in substantia nigra ipsilateral to the lesion. Computerized image analysis of sections from in situ hybridization revealed an increase in the number of silver grains over GAD mRNA positive neurons in the dorsolateral substantia nigra reticulata ipsilateral to the lesion. However, no change was observed in the ventromedial part suggesting that GAD mRNA expression in this part of the nigra is less sensitive to inhibition by caudate-putamen afferents. In agreement with in situ experiments, RNA blots showed a 2-fold increased level of GAD mRNA in substantia nigra ipsilateral to the lesion. The increased GAD mRNA expression in the deafferented substantia nigra suggests a disinhibition of nigral GABA neurons, resulting in an increased utilization of GABA in these substantia nigra neurons.     

Differential expression of neurotrophin and neurotrophin receptor mRNAs in and adjacent to fetal midbrain grafts implanted into the dopamine-denervated striatum

This study examined the expression of neurotrophins and neurotrophin receptors in the lesion/transplanted striatum at four different time points after transplantation. The ventral mesencephalic region was dissected from a single rat fetus at embryonic day 14 (E14) and implanted into the denervated striatum of rats with unilateral 6-hydroxydopamine lesions. Transplanted rats were killed at 1, 2, 3, or 4 weeks after transplantation surgery and the brains subsequently prepared for semiquantitative in situ hybridization analysis of neurotrophin and neurotrophin trk receptors. Hybridization of cRNA probes for trkB or trkC showed a time-dependent reduction within the transplant during the first 4 weeks after transplantation; hybridization of brain-derived neurotrophic factor or tyrosine hydroxylase mRNA probes within the transplant did not change significantly during the same posttransplantation period. Hybridization of the trkB mRNA probe in host striatum adjacent to the transplant was significantly higher than probe hybridization in the corresponding region of the intact striatum during the first 2 weeks after transplantation, but by the 3rd and 4th week, probe hybridization in the denervated/transplanted and intact striatum were the same. Lesioned animals without transplants maintained higher trkB mRNA probe hybridization in the denervated striatum than in the intact striatum at the same postlesion time points suggesting that lesioned/transplanted animals show a normalization of trkB mRNA probe hybridization. Hybridization of the trkC mRNA probe in the lesioned/transplanted striatum was significantly lower than that observed in the intact striatum 4 weeks after transplantation; however, at this same time point we observed a similar reduction of trkC probed hybridization in lesioned animals without transplants. The results of the study show dynamic neurotrophic activity occurring within the transplant and host tissue during the first month of transplant development.     

Developmental study of nerve growth factor receptor mRNA expression in the postnatal rat cerebellum

Nerve growth factor receptor (NGFR) mRNA expression was examined in the developing rat cerebellum with in situ hybridization histochemistry. The probe used in this study was the 57-mer oligonucleotide complementary to the rat NGFR cDNA (bases 798-855). The specificity of the hybridization was determined with a 'sense' probe. From the age of postnatal day 0 (PND 0), hybridization signals were detected in the external granule cell layer, Purkinje cell layer, and meningeal cells of the cerebellum. Until PND 10, the external granular cell layer consistently expressed high levels of NGFR message, with the densest hybridization signal observed in the proliferative zone. NGFR mRNA level in this layer rapidly decreased at later ages and was barely detectable after PND 16. In the Purkinje cell layer beyond PND 4, the hybridization signal became more intense, reached a peak level around PND 10, and then gradually decreased. NGFR mRNA was detected until PND 30 in this layer. Meningeal cells expressed NGFR message from PND 0 to PND 8. These results suggest that NGF may be involved in cerebellar development as a mediator of morphogenesis and/or synaptogenesis.     

Role of Dopamine in the Plasticity of Glutamic Acid Decarboxylase Messenger RNA in the Rat Frontal Cortex and the Nucleus Accumbens

The modulatory role of dopamine (DA) on the expression of mRNA encoding the large isoform of glutamic acid decarboxylase (GAD67), the biosynthesis enzyme of gamma aminobutyric acid (GABA), was examined in GABA neurons of two structures innervated by DA neurons originating from the ventral tegmental area (VTA): the medial frontal cortex (MFC) and the nucleus accumbens (NAcc). A bilateral electrolytic lesion of VTA was performed in rats to produce a DA denervation of both the MFC and NAcc. The efficacy of VTA lesions was verified by measurement of locomotor activity and by immunohistochemical detection of tyrosine hydroxylase in the mesencephalon. GAD67 mRNA was detected by in situ hybridization histochemistry using a ³⁵S-labelled cDNA probe. Densitometric analysis of GAD67 mRNA hybridization signals revealed in VTA-lesioned rats a significant decrease (-24%) in GAD67 mRNA levels in the prelimbic area of the MFC and no significant effect in the anterior cingulate area or the frontoparietal cortex. Single cell analyses by computer-assisted grain counting showed that the decrease in GAD67 mRNA levels in prelimbic MFC was due to a change in GAD67 mRNA expression in a subpopulation of GABA interneurons located in the deep cortical layers (V-VI). By contrast, in the NAcc of VTA-lesioned rats, GAD67 mRNA levels were significantly increased in the anterior part and in the core but were unchanged in the shell part. These results suggest that in two target structures of VTA DA neurons, GAD67 mRNA expression is, in normal conditions, under a tonic stimulatory and a tonic inhibitory DA control in the MFC and the NAcc respectively. A schematic diagram is proposed for functional interactions between these structures.     

Distribution of mRNA for CCK-B receptor in the brain of Mastomys natalensis: abundant expression in telencephalic neurons

The distribution of chelecystokinin B (CCK-B) receptors in the Mastomys brain was studied using Northern blot analysis and in situ hybridization technique. By Northern blot analysis using³²P-labeled cDNA probe, the cortex had the highest hybridization signal of CCK-B receptor mRNA in the brain. The olfactory bulb and hippocampus showed a moderate level of signals. In situ hybridization using³⁵S-labeled cRNA probes revealed a wide and region-specific distribution of CCK-B receptor mRNA in the telencephalon. Throughout the cerebral cortex, labeled cells were found in all layers, with higher intensities in layers II, V and VI. Pyramidal cells of the layer II of the piriform cortex showed the highest level of signals in the brain. In the hippocampus, most of the pyramidal cells of the Ammon's horn were labeled, although labeled cells were not detected in other layers. Distinct signals were also detected in the various amygdaloid nuclei, caudate-putamen, reticular thalamic nucleus, hypothalamic ventromedial nucleus and inferior colliculus. This distribution pattern may further support the prominent existence of CCK-B receptors in the brain particularly in the telencephalon.     

Developing patterns of nitric oxide synthesizing neurons in the rat striatum: histochemical analysis

The prenatal and postnatal development of NADPH-diaphorase (NADPH-d)/neuronal nitric oxide synthase (nNOS) positive neurons was studied in the striatum of rats. NADPH-d was demonstrated enzyme histochemically and nNOS immunohistochemically using a polyclonal antibody. NADPH-d neurons appeared in the ventrolateral part of the striatum on embryonic day 18 (E18). Thereafter, the number of NADPH-d neurons increased and began to distribute homogeneously in the striatum. The density of NADPH-d neurons became highest at postnatal day 5 (P5) and then decreased as the volume of the striatum continued to increase. The number of NADPH-d neurons reached its peak around 3-4 weeks after birth. The sizes of NADPH-d neurons were measured. The NADPH-d neurons grew larger until P14 (mean area 260 microm(2)) and became smaller thereafter (mean area 170 microm(2)). Patches of high NADPH-d activity and tyrosine hydroxylase (TH) immunoreactivity were also examined in the developing striatum. The distributions of NADPH-d patches overlapped with those of TH-immunoreactive patches by P10. The spatiotemporal appearance of nNOS and overlapping of nNOS patchy distribution with TH point to an important role of NO and to an interaction between nNOS and DA fibers during development of the striatum.     

Stability of GABAA/benzodiazepine receptor alpha 1 subunit mRNA expression in reeler mouse cerebellar Purkinje cells during postnatal development.

A [35S]cRNA probe was used for the visualization of GABAA/benzodiazepine (GABAA/BZ) receptor alpha 1 subunit mRNA in developing reeler mutant mouse cerebellum. A clear hybridization signal was observed throughout the malformed reeler cerebellum from birth. Labeling was associated with Purkinje cell bodies located in three subcortical masses. Additional labeled Purkinje cells were observed within the granule cell layer and at their normal position at the interface between the molecular and granule cell layers. All reeler Purkinje cells had comparable levels of grain density, regardless of their location within the cerebellar cortex. These results indicate that Purkinje cell malpositioning, and the resulting absence of a major complement of afferents throughout development, does not impair the expression of mRNA coding for the alpha 1 subunit of the GABAA/BZ receptor.     

Glutamate decarboxylase (GAD67 and GAD65) gene expression is increased in a subpopulation of neurons in the putamen of parkinsonian monkeys

The cellular distribution of the mRNAs encoding for the two isoforms of glutamate decarboxylase, GAD67 and GAD65, was analyzed by in situ hybridization histochemistry in the caudate nucleus and putamen of control and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonian squirrel monkeys. On brain sections processed with a radioactive and a digoxigenin-labeled cRNA probe, the GAD67 and GAD65 mRNAs were colocalized in virtually all labeled neurons of the caudate nucleus and putamen, in both control and MPTP-treated monkeys. Furthermore, neurons labeled with the GAD cRNAs constituted at least 90% of all striatal neurons, as estimated on adjacent Nissl-stained sections. In the two groups of monkeys, double-labeling experiments using a combination of radioactive GAD67 or GAD65 and digoxigenin-labeled preproenkephalin (PPE) cRNA probes showed that roughly half of all neurons labeled with the GAD cRNAs were also labeled with the PPE cRNA probe. When compared to controls, GAD67 and GAD65 mRNA levels were higher in the putamen, and to a lesser extent in the caudate nucleus, of MPTP-treated monkeys. Further analysis of labeling at the cellular level in a dorsolateral sector of the putamen revealed that GAD67 and GAD65 mRNA levels in MPTP-treated monkeys were increased in PPE-labeled (presumed striato-pallidal) neurons but not in PPE-unlabeled (presumed striato-nigral) neurons. Our results demonstrate that most neurons in the caudate nucleus and putamen of squirrel monkeys contain the mRNAs encoding for the two GAD isoforms. In addition, the selective increase in GAD mRNA levels in PPE-labeled neurons provides further evidence that striato-pallidal GABAergic neurons are hyperactive in MPTP-treated parkinsonian monkeys. Synapse 27:122–132, 1997. © 1997 Wiley-Liss, Inc.     

Kainic acid-induced seizures stimulate increased expression of nerve growth factor mRNA in rat hippocampus.

The influence of kainic acid (KA)-induced limbic seizure activity on the expression of mRNA for nerve growth factor (NGF) in adult rat brain was studied using in situ hybridization and S1 nuclease protection techniques with RNA probes complementary to murine and rat NGF mRNA. Within hippocampus, intracerebroventricular injection of 0.5 microgram KA caused a dramatic bilateral increase in hybridization of the 35S-labeled cRNA within stratum granulosum. This increase was first evident 1 h post-KA, appeared maximal at approximately 20-fold control levels at 2-3 h post-injection, and declined to control levels by 48 h post-injection. During the period of maximal hybridization, all but the deepest cells within stratum granulosum appeared to be autoradiographically labeled. Hybridization of the NGF cRNA probe was also increased within superficial layers of piriform and entorhinal cortex and, to much lesser extent, within scattered neurons of layers II and III of neocortex in KA-treated rats. In olfactory cortical areas, hybridization was maximally elevated 15.5-24.5 h after KA injection. In contrast to these effects, KA treatment did not consistently influence the density of hybridization, or number of neurons labeled, within the dentate gyrus hilus or the hippocampus proper (CA1-CA3). In agreement with the in situ hybridization results, S1 nuclease protection assay detected KA-induced increases in hybridization within pooled dentate gyrus/CA1 samples, but not hippocampal CA3 samples. These data support the conclusion that seizure activity stimulates a transient increase in NGF expression by select populations of forebrain neurons and indicates that experimental seizure paradigms might be further exploited for analyses of the mechanisms of NGF regulation and processing in the adult brain.     

NMDA receptor expression in the mouse cerebellar cortex

A detailed, light microscopic study on the distribution of the N-methyl-D-aspartate receptor subunit 1(NMDAR1) was carried out with immunohistochemistry and in situ hybridization on the cerebellar cortex of the mouse. With a monoclonal antibody, labeling of Purkinje cell bodies varied from intense to negative, while heavy dendritic staining was limited to the proximal dendrites (unlike the rat, which also had heavily stained distal dendrites). In the granular layer, the cell bodies and the dendritic shafts of Golgi II cells were only moderately stained, but very intense labeling was associated with granule cell bodies, and with their dendrites and dendritic endings in the glomeruli. The mossy and climbing fibers were negative. In situ hybridization with a cRNA probe showed levels and spatial distributions of NMDAR1 mRNA consistent with the immunolabeling pattern, in that signals were strongest in the granular and Purkinje cell layers and relatively low or absent in the molecular layer and white matter. The findings are consistent with the hypothesis that NMDAR1 may be especially well concentrated at the synaptic target sites of the mossy and climbing fibers. In the mouse, NMDAR1 at the parallel fiber sites associated with Purkinje cell spiny branchlets may differ from the rat in its level of expression or in its molecular configuration. © 1995 Wiley-Liss, Inc.     

Expression of basic fibroblast growth factor receptor messenger RNA in the periinfarcted brain tissue

The present study examined whether expression of basic fibroblast growth factor receptor (bFGFR) messenger ribonucleic acid (mRNA) was upregulated by focal ischemia. We have studied the in situ hybridization autoradiography for bFGFR mRNA in the rat model of middle cerebral artery (MCA) occlusion. Male Wistar rats were used for occlusion of the left MCA, and were sacrificed 1, 3, 7 and 14 days after MCA occlusion. In situ hybridization was performed on the brain sections of these animals and sham controls by using 35S-labeled antisense and sense (control) RNA probes for rat bFGFR. Expression of bFGFR mRNA was observed in the periinfarcted area of the rats within 1-14 days after MCA occlusion. Expression was evident in the whole hemisphere of the infarcted side, especially at 1 and 3 days after ischemia, but no expression was detected in the contralateral side. On microautoradiograms, the signals of bFGFR mRNA were detected in both neurons and non-neural cells located in the periinfarcted area. Upregulation of bFGFR mRNA detected in the periinfarcted brain tissue suggests that receptor-mediated action of bFGF may be related to preservation of neurons injured by ischemia.     

Effects of nigrostriatal lesions on the levels of messenger RNAs encoding two isoforms of glutamate decarboxylase in the globus pallidus and entopeduncular nucleus of the rat.

The neurotransmitter gamma-aminobutyric acid (GABA) is present in efferent neurons of the striatum and of the pallidum, one of the main striatal target areas. Dopaminergic nigrostriatal neurons play a critical role in the regulation of GABAergic neurotransmission in the striatum. In the present study, we investigated their role in the regulation of glutamate-decarboxylase (GAD) mRNA expression in two divisions of the pallidum in rats: the globus pallidus and entopeduncular nucleus, equivalent to the external and internal pallidum, respectively, of primates. Dopaminergic neurons were lesioned by unilateral injections of 6-hydroxydopamine (6-OHDA) in the substantia nigra of adult rats. Two or 3 weeks after the lesion, frontal cryostat-cut sections of the brain were processed for in situ hybridization histochemistry with 35S-labeled RNA probes synthesized from cDNAs encoding two distinct isoforms of GAD of respective molecular weight 67,000 (GAD67) and 65,000 (GAD65). The number of labeled cells was determined, and intensity of labeling in individual cells was analyzed by computerized image analysis on emulsion radioautographs. In the globus pallidus, the number of labeled neurons and intensity of labeling per cell were increased on the side ipsilateral to the lesion as compared with control rats in sections hybridized with the GAD67 RNA probe. No changes were detected on the side contralateral to the lesion or in the levels of labeling for GAD65 mRNA. Confirming previous data, the level of labeling for GAD65 mRNA was much higher than for GAD67 mRNA in the entopeduncular nucleus of control rats. In rats with a 6-OHDA lesion, labeling for both GAD67 and GAD65 mRNAs was decreased on the side contralateral, but not ipsilateral, to the lesion, as compared with control rats. The results show that lesions of the nigrostriatal pathway in rats affect the levels of mRNAs encoding two distinct isoforms of GAD in neurons of the globus pallidus and entopeduncular nucleus differently. In addition, results in the entopeduncular nucleus further support a bilateral effect of unilateral dopaminergic lesions.     

Coordinated upregulation of α1- and β11-tubulin mRNAs during collateral axonal sprouting of central peptidergic neurons

An in situ hybridization study was performed to determine the relationship between levels of mRNAs for the axonal growth-associated αl-tubulin and βZII-tubulin isotypes and the process of collateral axonal sprouting by identified central nervous system (CNS) neurons. A unilateral hypothalamic knife-cut was used to hemisect the hypothalarnoneurohypophysial tract, which results in a robust collateral sprouting response by the uninjured neurons of the contralateral supraoptic nucleus (SON) (Watt and Paden: Exp Neurol 111:9-24, 1991). At 10 and 30-35 days after the lesion, cryosections of the SON were obtained and hybridized with ³⁵S-labeled cDNA probeses specific to αl- and β11-tubulin mRNAs. Quantitative evaluation of the resulting autoradiographs revealed that αl-tubulin mRNA levels were significantly increased by 10 days in SON neurons that were undergoing collateral sprouting compared to controls and that this increase was sustained at 30-35 days post-lesion. Less marked increases in hybridization intensity of the βII-tubulin probe were also apparent in sprouting neurons at both 10 and 30-35 days after the lesion, but were statistically significant only at 10 days. The measured increases in intensity of hybridization of αl- and β11-tubulin probes are likely to be conservative estimates of the underlying increase in αl- and β11-tubulin mRNA levels because sprouting SON neurons undergo significant hypertrophy. High levels of both αl- and βII-tubulin mRNAs were also seen in surviving axotomized SON neurons ipsilateral to the hypothalamic lesion. We conclude that the pattern of regulation of αl- and β11-tubulin mRNAs in CNS neurons which are capable of supporting new axonal growth includes three elements: maintenance of significant basal αl- and β11-tubulin mRNA pools in mature neurons, rapid increases in the pool size of the mRNAs following stimulation of collateral sprouting, and sustained elevation of mRNA levels during the period of axonal sprouting. © 1995 Wiley-Liss, Inc.