Effects of neonatal rearing by different types of foster mother on the distribution of corticotropin-releasing factor neurons in the central amygdaloid nucleus in rats.

The mother-child relationship of newborns plays an essential role in the development of the central nervous system, and an inadequate relationship, such as mother-child separation, can cause deficits of mental function in adulthood. However, insufficient research has examined the effects of foster mothers. We assigned some neonatal rats to one of two foster mothers: one that was lactating and feeding her first litter (FL group) and one that had one previous experience of childbirth and feeding but no current litter (FE group). Other pups were raised by their own mother (OM group) or subjected to maternal separation (MS group). Pups were placed with the foster mother (FL and FE groups) or separated from their mother (MS group) for 3 h/day on postnatal days 1-20. At age 6 weeks, each group was divided into two subgroups, one with 30 min of acute restraint stress loading (FL-R, FE-R, OM-R, and MS-R) and one without it (FL, FE, OM, and MS). Then, we compared the density of corticotropin-releasing factor-immunoreactive (CRF-ir) neurons in the central amygdaloid nucleus (CeA). The density of CRF-ir neurons in the CeA was significantly lower in the FL-R and MS-R subgroups than in the FL and MS subgroups, respectively. The results suggest that differences in care received during the neonatal period affect maturation of CRF neurons in the CeA and may have negative effects on the synthesis and release of CRF.

Correlation of TGN-020 with the analgesic effects via ERK pathway activation after chronic constriction injury.

Extracellular regulated protein kinase (ERK) pathway activation in astrocytes and neurons has been reported to be critical for neuropathic pain development after chronic constriction injury. TGN-020 was found to be the most potent aquaporin 4 inhibitor among the agents studied. The present study aimed to assess whether the inhibition of aquaporin 4 had an analgesic effect on neuropathic pain and whether the inhibition of astrocytic activation and ERK pathway was involved in the analgesic effect of TGN-020. We thus found that TGN-020 upregulated the threshold of thermal and mechanical allodynia, downregulated the expression of interleukin-1ß, interleukin-6, and tumor necrosis factor-α, attenuated the astrocytic activation and suppressed the activation of mitogen-activated protein kinase pathways in the spinal dorsal horn and dorsal root ganglion. Additionally, TGN-020 suppressed ERK phosphorylation in astrocytes and neurons after injury. The findings suggested that the analgesic effects of TGN-020 in neuropathic pain were mediated mainly by the downregulation of chronic constriction injury-induced astrocytic activation and inflammation, which is via the inhibition of ERK pathway in the spinal dorsal horn and dorsal root ganglion.

Interactions between Sirt1 and MAPKs regulate astrocyte activation induced by brain injury in vitro and in vivo.

BACKGROUND: Astrocyte activation is a hallmark of traumatic brain injury resulting in neurological dysfunction or death for an overproduction of inflammatory cytokines and glial scar formation. Both the silent mating type information (Sirt1) expression and mitogen-activated protein kinase (MAPK) signal pathway activation represent a promising therapeutic target for several models of neurodegenerative diseases. We investigated the potential effects of Sirt1 upregulation and MAPK pathway pharmacological inhibition on astrocyte activation in vitro and in vivo. Moreover, we attempted to confirm the underlying interactions between Sirt1 and MAPK pathways in astrocyte activation after brain injury. METHODS: The present study employs an interleukin-1ß (IL-1ß) stimulated primary cortical astrocyte model in vitro and a nigrostriatal pathway injury model in vivo to mimic the astrocyte activation induced by traumatic brain injury. The activation of GFAP, Sirt1, and MAPK pathways were detected by Western blot; astrocyte morphological hypertrophy was assessed using immunofluorescence staining; in order to explore the neuroprotective effect of regulation Sirt1 expression and MAPK pathway activation, the motor and neurological function tests were assessed after injury. RESULTS: GFAP level and morphological hypertrophy of astrocytes are elevated after injury in vitro or in vivo. Furthermore, the expressions of phosphorylated extracellular regulated protein kinases (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), and phosphorylated p38 activation (p-p38) are upregulated, but the Sirt1 expression is downregulated. Overexpression of Sirt1 significantly increases the p-ERK expression and reduces the p-JNK and p-p38 expressions. Inhibition of ERK, JNK, or p38 activation respectively with their inhibitors significantly elevated the Sirt1 expression and attenuated the astrocyte activation. Both the overproduction of Sirt1 and inhibition of ERK, JNK, or p38 activation can alleviate the astrocyte activation, thereby improving the neurobehavioral function according to the modified neurological severity scores (mNSS) and balance latency test. CONCLUSIONS: Thus, Sirt1 plays a protective role against astrocyte activation, which may be associated with the regulation of the MAPK pathway activation induced by brain injury in vitro and in vivo.

Synaptic contact between median preoptic neurons and subfornical organ neurons projecting to the paraventricular hypothalamic nucleus.

It is known that the median preoptic nucleus (POMe) sends dense projections to the subfornical organ (SFO). However, the functional significance of these projections have not been well discussed. In this electron microscopic study, we investigated the presence of synapses between POMe-derived axon terminals and SFO neurons that project to the paraventricular hypothalamic nucleus (PVN). After injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex, into the PVN, many labeled neurons were found in the SFO. In contrast, after injection of an anterograde tracer, biotinylated dextran amine, in the POMe, abundant labeled axon varicosities were observed in the SFO. Using electron microscopy, synapses were identified between retrogradely labeled dendrites and cell bodies, and anterogradely labeled axon terminals, indicating that POMe neurons innervate SFO neurons projecting to the PVN. The possibility that POMe neurons play multiple roles in the neuronal circuit responsible for vasopressin release and/or cardiovascular regulation is also discussed.

LMTK1/AATYK1 is a novel regulator of axonal outgrowth that acts via Rab11 in a Cdk5-dependent manner.

Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1-deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WT or the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus, LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5-LMTK1-Rab11 is a novel signaling pathway involved in axonal outgrowth.

A DEAD-box RNA helicase Ddx54 protein in oligodendrocytes is indispensable for myelination in the central nervous system.

We recently reported that a new monoclonal antibody, 4F2, which labels oligodendroglial lineage cells, recognizes a DEAD-box RNA helicase Ddx54 and that Ddx54 binds to myelin basic protein (MBP) in brain and cultured oligodendrocytes. To elucidate the biological function of Ddx54, we generated a recombinant adenovirus, Ad-shRNA:Ddx54, expressing a short hairpin RNA to silence endogenous Ddx54 protein. The virus was intraventricularly injected into the brains of mice on postnatal day (PD) 2. The brains at PD 9 were then analyzed by immunohistochemistry. In untreated normal brain sections, as well as control brains that had been injected with Ad-ß-Gal, myelination of axons occurred in the corpus callosum with filamentous patterns of immunosignals of myelin-associated glycoprotein (MAG) and MBP. In Ad-shRNA:Ddx54-injected brain, substantial amounts of MAG and MBP immunosignals were present, but MBP immunosignals accumulated in the subplate layer and did not intrude into the emerging white matter. Immunoblot analysis revealed that Ddx54 knockdown caused a significant decrease in the level of 21.5 kDa MBP isoform and Ddx54, but the amount of Olig2; 2',3'-cyclic nucleotide 3' phosphodiesterase; MAG; three MBP isoforms (14, 17.5, and 18 kDa); and QKI-5, QKI-6, and QKI-7 proteins remained unchanged. Transfection of the Ddx54 expression vector into luciferase reporter-introduced neuroepithelial cells resulted in upregulated MBP promoter activity. Immunoprecipitation of Ddx54 protein in MBP-transfected HEK293 cells indicated that Ddx54 may directly interact with MBP mRNA. These results suggest that Ddx54 protein play an important role in central nervous system myelination, presumably in myelin sheath formation after the differentiation of oligodendrocytes.

Role of the lesion scar in the response to damage and repair of the central nervous system.

Traumatic damage to the central nervous system (CNS) destroys the blood-brain barrier (BBB) and provokes the invasion of hematogenous cells into the neural tissue. Invading leukocytes, macrophages and lymphocytes secrete various cytokines that induce an inflammatory reaction in the injured CNS and result in local neural degeneration, formation of a cystic cavity and activation of glial cells around the lesion site. As a consequence of these processes, two types of scarring tissue are formed in the lesion site. One is a glial scar that consists in reactive astrocytes, reactive microglia and glial precursor cells. The other is a fibrotic scar formed by fibroblasts, which have invaded the lesion site from adjacent meningeal and perivascular cells. At the interface, the reactive astrocytes and the fibroblasts interact to form an organized tissue, the glia limitans. The astrocytic reaction has a protective role by reconstituting the BBB, preventing neuronal degeneration and limiting the spread of damage. While much attention has been paid to the inhibitory effects of the astrocytic component of the scars on axon regeneration, this review will cover a number of recent studies in which manipulations of the fibroblastic component of the scar by reagents, such as blockers of collagen synthesis have been found to be beneficial for axon regeneration. To what extent these changes in the fibroblasts act via subsequent downstream actions on the astrocytes remains for future investigation.

Small molecule inhibitor of type I transforming growth factor-ß receptor kinase ameliorates the inhibitory milieu in injured brain and promotes regeneration of nigrostriatal dopaminergic axons.

Transforming growth factor-ß (TGF-ß), a multifunctional cytokine, plays a crucial role in wound healing in the damaged central nervous system. To examine effects of the TGF-ß signaling inhibition on formation of scar tissue and axonal regeneration, the small molecule inhibitor of type I TGF-ß receptor kinase LY-364947 was continuously infused in the lesion site of mouse brain after a unilateral transection of the nigrostriatal dopaminergic pathway. At 2 weeks after injury, the fibrotic scar comprising extracellular matrix molecules including fibronectin, type IV collagen, and chondroitin sulfate proteoglycans was formed in the lesion center, and reactive astrocytes were increased around the fibrotic scar. In the brain injured and infused with LY-364947, fibrotic scar formation was suppressed and decreased numbers of reactive astrocytes occupied the lesion site. Although leukocytes and serum IgG were observed within the fibrotic scar in the injured brain, they were almost absent in the injured and LY-364947-treated brain. At 2 weeks after injury, tyrosine hydroxylase (TH)-immunoreactive fibers barely extended beyond the fibrotic scar in the injured brain, but numerous TH-immunoreactive fibers regenerated over the lesion site in the LY-364947-treated brain. These results indicate that inhibition of TGF-ß signaling suppresses formation of the fibrotic scar and creates a permissive environment for axonal regeneration.

An in vitro model of the inhibition of axon growth in the lesion scar formed after central nervous system injury.

After central nervous system (CNS) injury, meningeal fibroblasts migrate in the lesion center to form a fibrotic scar which is surrounded by end feet of reactive astrocytes. The fibrotic scar expresses various axonal growth-inhibitory molecules and creates a major impediment for axonal regeneration. We developed an in vitro model of the scar using coculture of cerebral astrocytes and meningeal fibroblasts by adding transforming growth factor-beta1 (TGF-beta1), a potent fibrogenic factor. Addition of TGF-beta1 to this coculture resulted in enhanced proliferation of fibroblasts and the formation of cell clusters which consisted of fibroblasts inside and surrounded by astrocytes. The cell cluster in culture densely accumulated the extracellular matrix molecules and axonal growth-inhibitory molecules similar to the fibrotic scar, and remarkably inhibited the neurite outgrowth of cerebellar neurons. Therefore, this culture system can be available to analyze the inhibitory property in the lesion site of CNS.

The transcriptional repressor RP58 is crucial for cell-division patterning and neuronal survival in the developing cortex.

The neocortex and the hippocampus comprise several specific layers containing distinct neurons that originate from progenitors at specific development times, under the control of an adequate cell-division patterning mechanism. Although many molecules are known to regulate this cell-division patterning process, its details are not well understood. Here, we show that, in the developing cerebral cortex, the RP58 transcription repressor protein was expressed both in postmitotic glutamatergic projection neurons and in their progenitor cells, but not in GABAergic interneurons. Targeted deletion of the RP58 gene led to dysplasia of the neocortex and of the hippocampus, reduction of the number of mature cortical neurons, and defects of laminar organization, which reflect abnormal neuronal migration within the cortical plate. We demonstrate an impairment of the cell-division patterning during the late embryonic stage and an enhancement of apoptosis of the postmitotic neurons in the RP58-deficient cortex. These results suggest that RP58 controls cell division of progenitor cells and regulates the survival of postmitotic cortical neurons.

Expression of transforming growth factor-beta receptors in meningeal fibroblasts of the injured mouse brain.

The fibrotic scar which is formed after traumatic damage of the central nervous system (CNS) is considered as a major impediment for axonal regeneration. In the process of the fibrotic scar formation, meningeal fibroblasts invade and proliferate in the lesion site to secrete extracellular matrix proteins, such as collagen and laminin. Thereafter, end feet of reactive astrocytes elaborate a glia limitans surrounding the fibrotic scar. Transforming growth factor-beta1 (TGF-beta1), a potent scar-inducing factor, which is upregulated after CNS injury, has been implicated in the formation of the fibrotic scar and glia limitans. In the present study, expression of receptors to TGF-beta1 was examined by in situ hybridization histochemistry in transcortical knife lesions of the striatum in the mouse brain in combination with immunofluorescent staining for fibroblasts and astrocytes. Type I and type II TGF-beta receptor mRNAs were barely detected in the intact brain and first found in meningeal cells near the lesion 1 day postinjury. Many cells expressing TGF-beta receptors were found around the lesion site 3 days postinjury, and some of them were immunoreactive for fibronectin. After 5 days postinjury, many fibroblasts migrated from the meninges to the lesion site formed the fibrotic scar, and most of them expressed TGF-beta receptors. In contrast, few of reactive astrocytes expressed the receptors throughout the postinjury period examined. These results indicate that meningeal fibroblasts not reactive astrocytes are a major target of TGF-beta1 that is upregulated after CNS injury.

Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica.

BACKGROUND: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. OBJECTIVE: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. METHODS: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. RESULTS: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. CONCLUSION: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

Regeneration of nigrostriatal dopaminergic axons after transplantation of olfactory ensheathing cells and fibroblasts prevents fibrotic scar formation at the lesion site.

The fibrotic scar formed after central nervous system injury has been considered an obstacle to axonal regeneration. The present study was designed to examine whether cell transplantation into a damaged central nervous system can reduce fibrotic scar formation and promote axonal regeneration. Nigrostriatal dopaminergic axons were unilaterally transected in rats and cultures of olfactory-ensheathing cells (OECs), and olfactory nerve fibroblasts were transplanted into the lesion site. In the absence of transplants, few tyrosine hydroxylase-immunoreactive axons extended across the lesion 2 weeks after the transection. Reactive astrocytes increased around the lesion, and a fibrotic scar containing type IV collagen deposits developed in the lesion center. The immunoreactivity of chondroitin sulfate side chains and core protein of NG2 proteoglycan increased in and around the lesion. One and 2 weeks after transection and simultaneous transplantation, dopaminergic axons regenerated across the transplanted tissues, which consisted of p75-immunoreactive OECs and fibronectin-immunoreactive fibroblasts. Reactive astrocytes and chondroitin sulfate immunoreactivity increased around the transplants, whereas the deposition of type IV collagen and fibrotic scar formation were completely prevented at the lesion site. Transplantation of meningeal fibroblasts similarly prevented the formation of the fibrotic scar, although its effect on regeneration was less potent than transplantation of OECs and olfactory nerve fibroblasts. The present results suggest that elimination of the inhibitory fibrotic scar is important for neural regeneration.

Collateral projections from the subfornical organ to the median preoptic nucleus and paraventricular hypothalamic nucleus in the rat.

It is morphologically demonstrated that the subfornical organ (SFO) projects to the paraventricular hypothalamic nucleus (PVN) and also projects to the nucleus preopticus medianus (POMe), a relay nucleus of indirect projections from the SFO to PVN. However, it remains unknown, whether or not SFO neurons project collaterally to the POMe and PVN. To confirm this, a double retrograde labeling method was performed on rats using two fluorescent tracers. One tracer (red-colored FluoSpheres: FSR) was injected into the POMe and the other (Fast Blue: FB) was injected into the unilateral PVN at the same time. As a result, many retrogradely labeled neurons were found in the entire SFO. Of these, some neurons showed both FSR and FB fluorescence. Double-labeled neurons were found in about 8.7% of FSR-labeled neurons and 15.5% of FB-labeled neurons. The existence of double-labeled neurons indicates that single neurons in the SFO project simultaneously to the POMe and PVN via collateral axon branches. The data suggest that there are complicated neuronal pathways originating from the SFO in regulating cardiovascular and body fluid homeostasis.

Spatial and temporal expression of RP58, a novel zinc finger transcriptional repressor, in mouse brain.

RP58, a novel zinc finger protein containing a POZ domain, is a sequence-specific transcriptional repressor. To understand the role of this protein, we examined RP58 gene expression in the developing mouse brain by quantitative polymerase chain reaction (PCR) and in situ hybridization. RP58 mRNA expression was detected at embryonic day (E) 10 in the neuroepithelium, and subsequently in the ventricular zones of the cerebral cortex in the E12 embryo. Strong expression was observed in the preplate in the cerebral cortex from this stage onward. High levels of expression continued to be detected in the cortical plate and subventricular zone of the neocortex, hippocampus, and parts of the amygdala, but not in the thalamus or striatum. These results suggest that RP58 plays a crucial role in neuronal proliferation, migration, and differentiation in the developing cerebral cortex. RP58 is also expressed in the adult mouse neocortex, hippocampus, parts of the amygdala, and granule cells in the cerebellum. Double in situ hybridization using GAD67 or VGLUT1 probes revealed that RP58 is expressed in glutamatergic excitatory neurons.

Defects in reciprocal projections between the thalamus and cerebral cortex in the early development of Fezl-deficient mice.

Fez-like (Fezl), the forebrain embryonic zinc finger-like protein, is a transcriptional repressor selectively expressed in the deep layers of the developing cortex. We examined the thalamocortical and corticofugal pathways in Fezl-deficient fetal mice by using immunohistochemistry and by axonal labeling with the lipophilic dyes DiI and DiA, with special attention to the spatiotemporal relation between thalamocortical and corticofugal axons. In normal mice, thalamic and cortical axons meet in the internal capsule between embryonic day (E) 13.5 and E14.5 and fasciculate with each other as they extend to their targets, the cortex and thalamus, respectively. In Fezl-deficient mice, most of the thalamic and cortical axons stop in the internal capsule and at the pallial-subpallial boundary at E14.5, respectively. This abnormality is transient, and the thalamic and cortical axons reach their targets at E15.5, although the number of thalamic axons is remarkably reduced in the cortical anlage. Double labeling with DiI and DiA demonstrated close apposition of the thalamic and cortical axons in the subpallium and pallium as well as in the external capsule of this mutant after E15.5. Because the expression of genes that define the pallial-subpallial boundary and guidance molecules of thalamocortical axons did not show remarkable changes in Fezl-deficient mice, abnormal formation of thalamocortical pathway in this mutant may be caused by the defect of axons of cortical efferent neurons that express Fezl.

Protective effect of resveratrol against nigrostriatal pathway injury in striatum via JNK pathway.

Nigrostriatal pathway injury is one of the traumatic brain injury models that usually lead to neurological dysfunction or neuron necrosis. Resveratrol-induced benefits have recently been demonstrated in several models of neuronal degeneration diseases. However, the protective properties of resveratrol against neurodegeneration have not been explored definitely. Thus, we employ the nigrostriatal pathway injury model to mimic the insults on the brain. Resveratrol decreased the p-ERK expression and increased the p-JNK expression compared to the DMSO group, but not alter the p38 MAPK proteins around the lesion site by Western blot. Prior to the injury, mice were infused with resveratrol intracerebroventricularly with or without JNK-IN-8, a specific c-JNK pathway inhibitor for JNK1, JNK2 and JNK4. The study assessed modified improved neurological function score (mNSS) and beam/walking test, the level of inflammatory cytokines IL-1ß, IL-6 and TNF-α, and striatal expression of Bax and Bcl-2 proteins associated with neuronal apoptosis. The results revealed that resveratrol exerted a neuroprotective effect as shown by the improved mNSS and beam latency, anti-inflammatory effects as indicated by the decreased level of IL-1ß, TNF-α and IL-6. Furthermore, resveratrol up-regulated the protein expression of p-JNK and Bcl-2, down-regulated the expression of Bax and the number of Fluoro-Jade C (FJC) positive neurons. However, these advantages of resveratrol were abolished by JNK-IN-8 treatment. Overall, we demonstrated that resveratrol treatment attenuates the nigrostriatal pathway injury-induced neuronal apoptosis and inflammation via activation of c-JNK signaling.

Differential response of arcuate proopiomelanocortin- and neuropeptide Y-containing neurons to the lesion produced by gold thioglucose administration.

The effect of gold thioglucose (GTG) administration on neurons containing feeding-related peptides in the hypothalamic arcuate nucleus was examined in mice. Intraperitoneal GTG injection increased the body weight and produced a hypothalamic lesion that extended from the ventral part of the ventromedial nucleus to the dorsal part of the arcuate nucleus. Neurons containing proopiomelanocortin (POMC) and neuropeptide Y (NPY) present in the dorsal part of the arcuate nucleus were destroyed by GTG. In addition, the peptide-containing fibers that extended from the remaining arcuate neurons were degenerated at the lesion site. The number of POMC-containing fibers in the paraventricular nucleus, dorsomedial nucleus, and lateral hypothalamus was found to have decreased significantly when examined at 2 days and 2 weeks after the GTG treatment. In contrast, the number of NPY-containing fibers in the lateral hypothalamus remained unchanged after the GTG treatment, probably because of the presence of an unaffected NPY-containing fiber pathway passing through the tuberal region and projecting onto the lateral hypothalamus. The number of NPY-immunoreactive fibers in the paraventricular and dorsomedial nuclei showed a moderate but significant decrease at 2 days after the GTG treatment, but it recovered to the normal levels 2 weeks later. The NPY-containing fibers were found to have regenerated across the lesion site 2 weeks later, and this might contribute to the recovery of the NPY-immunoreactive fibers in these regions. The present results first demonstrate that POMC- and NPY-containing neurons in the arcuate nucleus respond differently to the lesion produced by the GTG treatment.

Particular subpopulations of midbrain and hypothalamic dopamine neurons express vesicular glutamate transporter 2 in the rat brain.

Vesicular glutamate transporters (VGLUT1, -2, and -3) mediate the accumulation of transmitter glutamate into synaptic vesicles in glutamatergic neurons. VGLUT1 and VGLUT2 are more reliable glutamatergic neuron markers, since VGLUT3 also exists in other neuron types. To study whether the dopaminergic neuron uses glutamate as a cotransmitter, we analyzed VGLUTs expression in dopamine neurons of adult male rats by in situ hybridization and immunohistochemistry. In the ventral midbrain, in situ hybridization analysis revealed no VGLUT1 mRNA expression, a widespread but discrete pattern of VGLUT2 mRNA expression, and a highly limited expression of VGLUT3 mRNA. Reverse-transcriptase polymerase chain reaction analysis detected full-length VGLUT2 gene transcripts in the ventral midbrain. Using in situ hybridization combined with tyrosine hydroxylase (TH) immunostaining, only VGLUT2 signals were detectable in some TH-labeled neurons of A10 dopamine neuron groups, with the highest incidence (20%) in the rostral linear nucleus of the ventral tegmental area. In the forebrain, VGLUT2 signals were demonstrated in half of the A11 TH-labeled neurons in the hypothalamus. Double-label immunostaining for VGLUT2 and vesicular monoamine transporter 2 or TH showed that double-labeled varicosities are rarely observed in any target regions examined of A10 and A11 dopamine neuron groups. These results indicate that VGLUT2 is expressed in subsets of A10 and A11 dopamine neurons, which might release dopamine and glutamate separately from different varicosities in the majority of their single axons.