FGF-2-mediated protection of cultured mesencephalic dopaminergic neurons against MPTP and MPP+: Specificity and impact of culture conditions,non-dopaminergic neurons,and astroglial cells

The protective role of basic fibroblast growth factor (FGF-2) for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and methylpyridiniumion (MPP⁺)-lesioned dopaminergic (DAergic) nigrostriatal neurons was studied, using dissociated cell cultures of embryonic day (E) 14 rat mesencephalon. Cells were grown in different culture media and received FGF-2 (5 ng/ml) and/or the toxins (5 μM) at various schedules, but were consistently allowed to differentiate for 3 days prior to becoming exposed to the toxin. Survival of tyrosine hydroxylase (TH)-immunoreactive cells at 7 days was only markedly impaired by MPTP, if horse serum (HS) or bovine serum albumine (BSA) were omitted from the culture medium. FGF-2 increased the number of TH-immunoreactive cells, and this increase not diminished by MPTP under any culture condition. Uptake of ³H-DA was significantly reduced by MPTP in HS- and BSA-containing, but not in protein-less cultures. A protective effect by FGF-2 was only seen in the presence of BSA. MPP⁺ caused a more pronounced reduction in ³H-DA uptake than MPTP, and this effect was partially reversed by the addition of FGF-2, unless cultures contained HS. Neurofilament protein (NF), an indirect measure for the total number of neurons present in the cultures, was not significantly reduced by MPTP or MPP⁺ corroborating the specificity of the toxin for DAergic neurons, which constitute only a minor fraction in these cultures. In line with the wide spectrum of target neurons of FGF-2, this factor significantly increased NF contents under any culture condition. Quantification of the amounts of glial fibrillary acidic protein (GFAP) revealed stimulatory effects of FGF-2 (2.5- to 4-fold) and at least 10-fold higher levels in the presence as compared to the absence of HS. These data show that FGF-2 can protect DAergic neurons against MPTP- and MPP⁺-mediated damage. However, the effects of the toxins as well as of FGF-2 are partially dependent on culture conditions. Variations in the effectiveness of toxins and FGF-2 are not overtly related to the total numbers of neurons or astroglial cells, but may reflect culture type-dependent alterations of neuronal and glial metabolism. © 1993 Wiley-Liss, Inc.     

Stereological analysis of Ca(2+)/calmodulin-dependent protein kinase II alpha -containing dorsal root ganglion neurons in the rat: colocalization with isolectin Griffonia simplicifolia,calcitonin gene-related peptide,or vanilloid receptor 1

The enzyme Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is widely distributed in the nervous system. A previous report describes immunostaining for CaMKII alpha in dorsal root ganglion (DRG) neurons. In this study, CaMKII alpha is colocalized in the rat with three putative markers of nociceptive DRG neurons, isolectin Griffonia simplicifolia (I-B4), identifying small-diameter, "peptide-poor" neurons; calcitonin gene-related peptide (CGRP), identifying " peptide-rich" neurons; or the vanilloid receptor 1 (VR1), identifying neurons activated by heat, acid, and capsaicin. Lumbar 4 and 5 DRG sections were labeled using immunofluorescence or lectin binding histochemistry, and percentages of single and double-labeled CaMKIIalpha neurons were determined. Stereological estimates of total neuron number in the L4 DRG were 13,815 +/- 2,798 and in the L5 DRG were 14,111 +/- 4,043. Percentages of single-labeled L4 DRG neurons were 41% +/- 2% CaMKII alpha, 38% +/- 3% I-B4, 44% +/- 3% CGRP, and 32% +/- 6% VR1. Percentages of single-labeled L5 DRG neurons were 44% +/- 5% CaMKII alpha, 48% +/- 2% I-B4, 41% +/- 7% CGRP, and 39% +/- 14% VR1. For L4 and L5, respectively, estimates of double-labeled CaMKII alpha neurons showed 34% +/- 2% and 38% +/- 17% labeled for I-B4, 25% +/- 14% and 19% +/- 10% labeled for CGRP, and 37% +/- 7% and 38% +/- 5% labeled for VR1. Conversely, for L4 and L5, respectively, 39% +/- 14% and 38% +/- 7% I-B4 binding neurons, 24% +/- 12% and 23% +/- 10% CGRP neurons, and 42% +/- 7% and 35% +/- 7% VR1 neurons labeled for CaMKIIalpha. The mean diameter of CaMKII alpha - labeled neurons was approximately 27 microm, confirming that this enzyme was preferentially localized in small DRG neurons. The results indicate that subpopulations of DRG neurons containing CaMKII alpha are likely to be involved in the processing of nociceptive information. Thus, this enzyme may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory neurons.     

Overexpression of Septin 4, the Drosophila homologue of human CDCrel-1, is toxic for dopaminergic neurons

parkin loss-of-function mutations are linked to autosomal recessive juvenile parkinsonism. Parkin is an E3 ubiquitin ligase that promotes degradation of specific target proteins by the proteasome. It has been proposed that loss of Parkin activity will result in accumulation of its substrates, thus leading to dopaminergic (DA) neuron death. In Drosophila, parkin mutations cause degeneration of a subset of DA neurons in the brain but no Parkin substrates have yet been described. Here we characterized the septin 4 gene, which encodes the Drosophila orthologue of human CDCrel-1, a Parkin substrate. We showed that Septin 4 overexpression causes age-dependent disruption of DA neuron integrity in the dorsomedial cluster, which is suppressed by coexpression of Parkin and enhanced by reducing parkin function. Furthermore, other phenotypes caused by Septin 4 overexpression are also enhanced in a heterozygous parkin mutant background. This indicates that Septin 4 accumulation is toxic for DA neurons and suggests that Septin 4 could be a genuine substrate of Drosophila Parkin. Regarding this, we also showed that both proteins are able to interact physically with each other in vitro, thus supporting this hypothesis.     

Pretreatment with antisense oligodeoxynucleotide against D(2) or D(3) receptor mRNA diminished dopamine's inhibitory effect on dorsomedial arcuate neurons in brain slices of estrogen-treated ovariectomized rats

A high percentage of dopamine (DA)-responsive neurons has been repeatedly shown in hypothalamic dorsomedial arcuate nucleus (dmARN) using single-unit recording in brain slices. Both D(2) and D(3) receptors may be involved in the inhibitory action of DA as indicated by results obtained from using specific DA agonists and antagonists. To further delineate the DA receptor types involved, ovariectomized, estrogen-primed Sprague-Dawley rats pretreated with antisense oligodeoxynucleotide (ODN, 10 microg/3 microl, i.c.v.) against either D(2) or D(3) receptor mRNA for 2 days were used in this study for brain slice preparation. Rats pretreated with aCSF, random antisense or sense ODNs were used as controls. DA (5-50 nmol) inhibited a majority of dmARN neurons in brain slices prepared from rats pretreated with aCSF (71.4% of 35 U), random antisense/sense ODN for D(2) (67.6%, n=34), D(3) (59.5%, n=42), or D(2) plus D(3) (60.5%, n=38) mRNAs. In contrast, DA inhibited 43.6% (n=39) of dmARN neurons in slices prepared from D(2), and 38.5% (n=39) from D(3) antisense ODN-pretreated rats. Furthermore, in brain slices prepared from rats pretreated with combined D(2) and D(3) antisense ODNs, DA only inhibited 18.4% (n=38) of dmARN neurons. We conclude that both D(2) and D(3) receptors are involved in the action of DA on dmARN neurons in ovariectomized, estrogen-treated rats.     

Effect of blood pressure on A2 noradrenergic neurons

Single-unit recordings of A2 noradrenergic neurons were obtained in urethane-anesthetized rats. Norepinephrine-induced elevations in arterial pressure produced small decreases in the discharge rate of the cells; this effect was not observed in animals with bilateral sections of the ninth and tenth nerves. The activity of A2 cells was neither pulse-synchronous at high levels of arterial pressure nor significantly affected by carotid stretch. It is concluded that these neurons do not receive a significant baroreceptor input.     

Effects of constant light and darkness on the intrapineal neurons of golden hamsters,stained for tyrosine hydroxylase A morphometric analysis

Summary Tyrosine hydroxylase (TH)-positive neurons (TH neurons) were found in the pineal gland of golden hamsters. To examine possible relations between TH neurons and environmental light, we kept male animals under constant light (LL) and darkness (DD) for a week, and morphometrically compared the number, size, and immunoreactivity of TH neurons with those of control animals kept under 12L/12D (LD), using an image processor, Nexus 6400. In LL animals, the number of TH neurons/mm² of pineal tissue and each cell area were decreased, and immunoreactivity to TH was less than in LD animals. In DD animals, the number of TH neurons and each cell area were increased, and immunoreactivity decreased slightly. These data suggested that environmental light affected the TH neurons, and the amount of TH in the neurons would be decreased by LL, but increased by DD.     

A survival motor neuron:tetanus toxin fragment C fusion protein for the targeted delivery of SMN protein to neurons

Spinal muscular atrophy (SMA) is a degenerative disorder of spinal motor neurons caused by homozygous mutations in the survival motor neuron (SMN1) gene. Because increased tissue levels of human SMN protein (hSMN) in transgenic mice reduce the motor neuron loss caused by murine SMN knockout, we engineered a recombinant SMN fusion protein to deliver exogenous hSMN to the cytosolic compartment of motor neurons. The fusion protein, SDT, is comprised of hSMN linked to the catalytic and transmembrane domains of diphtheria toxin (DTx) followed by fragment C of tetanus toxin (TTC). Following overexpression in Escherichia coli, SDT possessed a subunit molecular weight of approximately 130 kDa as revealed by both SDS-PAGE and immunoblot analyses with anti-SMN, anti-DTx, and anti-TTC antibodies. Like wild-type SMN, purified SDT showed specific binding in vitro to an RG peptide derived from Ewing's sarcoma protein. The fusion protein also bound to cultured primary neurons in amounts similar to those achieved by TTC. Unlike the case with TTC, however, immunolabeling of SDT-treated neurons with anti-TTC and anti-SMN antibodies showed staining restricted to the cell surface. Results from cytotoxicity studies in which the DTx catalytic domain of SDT was used as a reporter protein for internalization and membrane translocation activity suggest that the SMN moiety of the fusion protein is interfering with one or both of these processes. While these studies indicate that SDT may not be useful for SMA therapy, the use of the TTC:DTx fusion construct to deliver other passenger proteins to the neuronal cytosol should not be ruled out.     

The 5-HT2A receptor is widely distributed in the rat spinal cord and mainly localized at the plasma membrane of postsynaptic neurons

Serotonin (5-HT) plays a major role at the spinal level by modulating most spinal functions through several receptor subtypes including the 5-HT2A receptor. To gain further insight into the cellular role of this receptor, we performed an immunocytochemical study of 5-HT2A receptors in the rat spinal cord, at light and electron microscope levels. The results showed that 5-HT2A receptors were widely distributed in the spinal cord at all segmental levels. Immunolabeling was particularly dense in lamina IX and in the dorsal horn lamina IIi. Immunoreactive cell bodies were numerous in lamina IX, where many but not all motoneurons were labeled, as shown by double labeling with choline acetyltransferase antibodies. Stained cell bodies were also observed in the gray matter. The study at the ultrastructural level focused on the lumbar dorsal horn (laminae I-II) and ventral horn (lamina IX). At both levels, 5-HT2A immunoreactivity was mainly postsynaptic on dendrites and cell bodies. However, a little presynaptic labeling was also observed in axon and axon terminals, some of them containing large granular vesicles attesting to their peptidergic nature. The main result of our study was the "nonsynaptic" plasma membrane localization of 5-HT2A receptors covering a large surface of cell bodies and dendrites, suggesting a paracrine form of action of serotonin. These observations are consistent with a double role (pre- and postsynaptic) for serotonin on these receptors on various cellular targets.     

A role for oligodendrocyte-derived IGF-1 in trophic support of cortical neurons

Neurons and glia interact in the development of mammalian central nervous systems and in the maintenance of stable myelinated axons. Recent evidence suggests a role for oligodendrocytes in providing trophic support for neurons during development and in the mature nervous system. This work prompted us to study oligodendrocyte influences on neuronal survival and death in vitro. Rat embryonic cortical neurons were co-cultured with purified oligodendrocytes at different developmental stages and separately with oligodendrocyte-conditioned medium. Neuronal survival was measured by immunocytochemistry and 3H-GABA uptake. Neurons show a marked increase in survival when co-cultured directly with oligodendrocyte precursors (OPCs) and differentiated oligodendrocytes. Neurons cultured in the presence of OPCs separated by a permeable membrane and those cultured in medium conditioned by oligodendrocytes also show a significant increase in survival. Medium conditioned by differentiated oligodendrocytes provides a greater survival effect than medium conditioned by OPCs. Neutralising antibodies to IGF-1, but not to other candidate trophic factors, block the soluble survival effect of oligodendrocytes. Cells of the oligodendrocyte lineage produce IGF-1 and recombinant IGF-1 promotes neuronal survival under identical conditions. This study provides evidence that OPCs and differentiated oligodendrocytes support neuronal survival by both contact-mediated and soluble mechanisms and that IGF-1 significantly contributes to this effect.     

Inhibition of feline spinal cord dorsal horn neurons following electrical stimulation of nucleus paragigantocellularis lateralis. A comparison with nucleus raphe magnus

The effect of electrical stimulation applied to the nucleus paragigantocellularis lateralis (PGL) was assessed on the somatosensory responses of functionally identified spinal cord dorsal horn neurons in the cat. Neurons were classified as low thershold mechanoreceptive, wide dynamic range or nociceptive specific. The responses of over 95% of all neurons tested were inhibited by a conditioning stimulus to the PGL. For each cell the threshold current intensity necessary to produce inhibition from the PGL (inhibitory threhold) was determined. Analysis of the incidence of inhibition and the inhibitory thresholds showed that the PGL-induced inhibition was not selective for a particular class of neuron. Due to the many similarities between the PGL and the nucleus raphe magnus (NRM), a comparison was made between each region's potency in inhibiting the responses of spinal cord neurons. Based on an analysis of inhibitory thresholds, the PGL was found to be significantly more potent than the NRM. These results indicate the PGL to be an important site from which descending modulation of spinal cord somesthetic information emanates.     

Preferential neurotrophic activity of fibroblast growth factor-20 for dopaminergic neurons through fibroblast growth factor receptor-1c

Degeneration of dopaminergic neurons of the substantia nigra causes Parkinson's disease. Therefore, neurotrophic factors for dopaminergic neurons are of substantial clinical interest. Fibroblast growth factor (FGF)-20 preferentially expressed in the substantia nigra pars compacta (SNPC) of the rat brain significantly enhanced the survival of midbrain dopaminergic neurons. Here we examined the mechanism of action of FGF-20 on dopaminergic neurons. FGF-20 slightly enhanced the survival of total neurons of the midbrain, indicating that it preferentially enhanced the survival of dopaminergic neurons. FGF receptor (FGFR)-1c was found to be expressed abundantly in dopaminergic neurons in the SNPC but at much lower levels in neurons of other midbrain regions by in situ hybridization. FGF-20 was also found to bind FGFR-1c with high affinity with the BIAcore system. Furthermore, FGF-20 activated the mitogen-activated protein kinase (MAPK) pathway, which is the major intracellular signaling pathway of FGFs. Both the FGFR-1 inhibitor SU5402 and the MAPK pathway inhibitor PD98059 also significantly inhibited the activation of the MAPK pathway by FGF-20 and the neurotrophic activity of FGF-20. The present findings indicate that the activation of the MAPK pathway by FGF-20 signaling through FGFR-1c plays important roles in the survival of dopaminergic neurons in the SNPC.     

神经元形态分类和命名的机制研究

The morphological and functional characteristics of neurons are quite varied and complex. There is a need for a comprehensive approach for distinguishing and classifying neurons. Similar to the biological species classification system, this study proposes a morphological classification system for neurons based on principal component analysis. Based on four principal components of neuronal morphology derived from principal component analysis, a nomenclature system for neurons was obtained. This system can accurately distinguish between the same type of neuron from different species.     

A role for tumor necrosis factor alpha in death of dopaminergic neurons following neural transplantation

Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease (PD). We have examined the temporal release profile of an inflammatory cytokine, tumor necrosis factor-alpha (TNFalpha), following transplantation of fetal mesencephalic tissue into the rat striatum. The amounts of TNFalpha released in vivo when added to cultures of embryonic DA neurons, significantly reduced the survival of DA neurons in vitro, and this cell death could be prevented by the inclusion of an antibody to the TNFalpha receptor type 1. Inclusion of this antibody in cell suspensions during transplantation also increased the survival of transplanted fetal DA neurons by approximately 250%. Use of this therapeutic antibody approach may offer significant improvements to neural transplantation as a treatment for PD.     

Interleukin-2 as a neurotrophic factor for supporting the survival of neurons cultured from various regions of fetal rat brain

Interleukin(IL)-2 supported the survival and enhanced neurite extension of cultured hippocampal neurons prepared from embryonic 18-day-old rats. This neurotrophic effect was observed at concentrations of 2 to 200 U/ml, and almost all the neurons could survive for more than 2 days in the presence of 200 U/ml of IL-2. This viability-promoting effect of IL-2 on the neurons was completely blocked with anti-IL-2 antibodies. IL-2 also supported the survival of cultured cortical, striatal, and septal neurons. These results indicate that IL-2 has a survival-promoting effect on a wide variety of neurons. On the other hand, IL-2 did not affect the choline acetyltransferase (ChAT) activity of striatal neurons, suggesting that this cytokine does not act as a differentiation factor for striatal cholinergic neurons. © 1993 Wiley-Liss, Inc.     

An electrophysiological study of the forebrain projection of nucleus commissuralis: Preliminary identification of presumed A2 catecholaminergic neurons

Using a double-labeling technique (HRP combined with catecholamine fluorescence), up to 80% of all CA-containing neurons visualized in the nucleus commissuralis were found to project to or through the median forebrain bundle area (MFB). In addition at least 90% of all nucleus commissuralis neurons projecting through the MFB were found to be catecholaminergic. In a series of chloral hydrate-anesthetized rats, nucleus commissuralis neurons projecting through the MFB were identified with single-unit recordings by antidromic (AD) activation. These cells had a conduction velocity of about 0.5 m/s and a firing rate of 0-14 spikes/s. The pattern of discharge of these neurons was not correlated with the heart rate; they were unaffected by single-unit stimulation applied to the sciatic nerve but were powerfully excited by vagus nerve stimulation. For comparative purposes, NE-containing neurons were also recorded in the locus coeruleus (A6) in the course of the same experiments; in contrast with MFB-activated commissuralis neurons, A6 neurons were excited by both visceral (vagus nerve) and somatic (sciatic nerve) stimulation. The spontaneous firing rate of MFB-activated commissuralis neurons was inhibited by the intravenous administration of a low dose of the centrally acting antihypertensive agent clonidine (ED50: 28 micrograms/kg).     

Immunocytochemical evidence for stimulatory control by the ventral noradrenergic bundle of parvocellular neurons of the paraventricular nucleus secreting corticotropin releasing hormone and vasopressin in rats

The regulation, by catecholaminergic innervation, of parvocellular neurons of the paraventricular nuclei (PVN) secreting corticotropin releasing hormone (CRH) and vasopressin (Vp) was studied by immunocytochemical visualization of both neurohormones in control rats and in rats given discrete injections of 6-hydroxydopamine in the ventral noradrenergic ascending bundle (VNAB). In both groups, the changes in immunostaining intensities observed in axon terminals of the external median eminence and in PVN perikarya 48 h after a blockade of axoplasmic transport by intraventricular injections of colchicine, served as an index for hormonal release and synthesis. In controls, this treatment induced a strong decrease in CRH and Vp immunoreactivity within the terminals, together with intense labeling of PVN perikarya containing CRH. By contrast, bilateral VNAB lesions strikingly inhibited both the colchicine-induced reduction of the CRH and Vp immunoreactivity in axons and the accumulation of CRH in the perikarya. Unilateral VNAB lesions induced similar alterations but these were restricted to the ipsilateral PVN and median eminence. Comparison of these immunocytochemical data with earlier physiological observations on the effects of VNAB lesions on ACTH secretion indicates that the catecholaminergic afferents to the PVN conveyed by the VNAB stimulate the release and the synthesis of CRH and Vp by parvocellular neurons projecting into the external median eminence.     

Defining the role of Drosophila lateral neurons in the control of circadian rhythms in motor activity and eclosion by targeted genetic ablation and PERIOD protein overexpression

The ventral lateral neurons (LNvs) of the Drosophila brain that express the period (per) and pigment dispersing factor (pdf) genes play a major role in the control of circadian activity rhythms. A new P-gal4 enhancer trap line is described that is mostly expressed in the LNvs This P-gal4 line was used to ablate the LNvs by using the pro-apoptosis gene bax, to stop PER protein oscillations by overexpressing per and to block synaptic transmission with the tetanus toxin light chain (TeTxLC). Genetic ablation of these clock cells leads to the loss of robust 24-h activity rhythms and reveals a phase advance in light-dark conditions as well as a weak short-period rhythm in constant darkness. This behavioural phenotype is similar to that described for disconnected1 (disco1) mutants, in which we show that the majority of the individuals have a reduced number of dorsally projecting lateral neurons which, however, fail to express PER. In both LNv-ablated and disco1 flies, PER cycles in the so-called dorsal neurons (DNs) of the superior protocerebrum, suggesting that the weak short-period rhythm could stem from these PDF-negative cells. The overexpression of per in LNs suppresses PER protein oscillations and leads to the disruption of both activity and eclosion rhythms, indicating that PER cycling in these cells is required for both of these rhythmic behaviours. Interestingly, flies overexpressing PER in the LNs do not show any weak short-period rhythms, although PER cycles in at least a fraction of the DNs, suggesting a dominant role of the LNs on the behavioural rhythms. Expression of TeTxLC in the LNvs does not impair activity rhythms, which indicates that the PDF-expressing neurons do not use synaptobrevin-dependent transmission to control these rhythms.     

Long-term activation of adenosine A(2a) receptors blocks glutamate excitotoxicity in cultures of avian retinal neurons

Previous work showed the presence of adenosine receptors as well as adenosine uptake and release mechanisms in developing chick retinal neurons in culture. In the present work we show that exogenous glutamate or kainate promotes extensive cell death in these cultures which is blocked when the cultures are previously incubated with adenosine. Addition of glutamate or kainate to purified cultures of retinal neurons and photoreceptors induced massive death of cultured cells which was inhibited in both cases by preincubation with MK801, an NMDA antagonist, or DNQX, an AMPA/kainate antagonist. Cell death was also greatly attenuated by preincubation with adenosine plus EHNA, an adenosine deaminase inhibitor, NBI, an adenosine uptake blocker, the permeable cAMP analogs 8-Br cAMP and Sp cAMP and the A_2a agonists CGS 21680 and DPMA, but not with the A₁ receptor agonist CHA. Kinetic studies performed determining the intracellular LDH activity showed that maximal death was observed after 8 h and in concentrations of glutamate as low as 50 μM. We also observed a time-dependent protective effect of adenosine beginning after 1 h of preincubation and reaching a maximal effect after 24 h, indicating its association with changes in cellular metabolism induced by long-term exposure of cells to the nucleoside. The results show that adenosine inhibits glutamate toxicity in retinal neurons through a long-term activation of A_2a receptors and elevation of intracellular cyclic AMP levels.     

A photoetched cell relocation matrix for long-term, quantitative observations of selected individual neurons in culture

A photoetched matrix of indium tin oxide (ITO) on glass has been developed and tested as a tool to assist in the relocation and identification of individual neuronal cells in culture. The matrix is formed by 10-15 micron wide and 300 A thick ITO lines which subdivide a 1-cm2 area into 625 smaller squares. Each of the smaller squares measures 400 micron on a side and contains a photoetched two-letter "address". The address code allows precise relocation of specific regions of a culture as well as verification of the identities of individual neurons selected for repeated observation. Marks at 50 micron intervals along the sides of the address squares permit quantitative analysis of morphological changes, cell migration, reaggregation, etc. The ITO is transparent and does not interfere with visualization of even fine details of cells with high power microscopy.     

A novel dopamine transporter transgenic mouse line for identification and purification of midbrain dopaminergic neurons reveals midbrain heterogeneity

Midbrain dopaminergic (DAergic) neurons are a heterogeneous cell group, composed of functionally distinct cell populations projecting to the basal ganglia, prefrontal cortex and limbic system. Despite their functional significance, the midbrain population of DAergic neurons is sparse, constituting only 20 000–30 000 neurons in mice, and development of novel tools to identify these cells is warranted. Here, a bacterial artificial chromosome mouse line [Dat1‐enhanced green fluorescent protein (eGFP)] from the Gene Expression Nervous System Atlas (GENSAT) that expresses eGFP under control of the dopamine transporter (DAT) promoter was characterized. Confocal microscopy analysis of brain sections showed strong eGFP signal reporter in midbrain regions and striatal terminals that co‐localized with the DAergic markers DAT and tyrosine hydroxylase (TH). Thorough quantification of co‐localization of the eGFP reporter signal with DAT and TH in the ventral midbrain showed that a vast majority of eGFP‐expressing neurons are DAergic. Importantly, expression profiles also revealed DAergic heterogeneity when comparing substantia nigra and ventral tegmental area. Dat1‐eGFP mice showed neither change in synaptosomal DA uptake nor altered levels of DAT and TH in both striatum and midbrain. No behavioural difference between Dat1‐eGFP and wild‐type was found, suggesting that the strain is not aberrant. Finally, cell populations highly enriched in DAergic neurons can be obtained from postnatal mice by fluorescence‐activated cell sorting and the sorted neurons can be cultured in vitro. The current investigation demonstrates that eGFP expression in this mouse line is selective for DAergic neurons, suggesting that the Dat1‐eGFP mouse strain constitutes a promising tool for delineating new aspects of DA biology.