EGFP-expressing Neurons Research Articles

Two Groups of eGFP-Expressing Neurons with Distinct Characteristics in the Neocortex of GIN Mice

GIN (GFP-expressing inhibitory interneuron) transgenic mice are believed to express the enhanced GFP (eGFP) in a subset of somatostatin (SST)-expressing interneurons in the neocortex and have been widely used in the study on SST interneurons. Previous studies showed that eGFP+ neurons in the neocortex are distributed in the layer II-IV and upper layer V (cortical eGFP neurons) and contain SST. In this study, we reported a new group of eGFP+ neurons in GIN mice at early postnatal ages, which was located in the deep layer of the lateral neocortex as clusters (cluster eGFP neurons). Cluster eGFP neurons were noticeable at birth but disappeared within two months, in contrast to cortical eGFP neurons that started to appear around postnatal day 3 to 5 and existed through life. Cluster eGFP neurons were not immunoreactive for SST antibodies, contrary to cortical eGFP neurons. They were also not immunolabeled by parvalbumin, a marker for another major type of interneurons, and Ca2+/calmodulin-dependent kinases II, a commonly used marker for excitatory neurons. Firing rate, afterhyperpolarization, and excitatory synaptic activity significantly enhanced in cortical eGFP neurons during postnatal development, but these properties remained mostly unchanged in cluster eGFP neurons. Short-term plasticity of the excitatory synapse showed robust facilitation in cortical eGFP neurons but depression in cluster eGFP neurons. These results implied that eGFP might also be expressed in other types of cortical neurons in addition to SST-containing interneurons in GIN mice at early postnatal ages.

Morphological and Physiological Plasticity of Spinal Lamina II GABA Neurons Is Induced by Sciatic Nerve Chronic Constriction Injury in Mice.

Mice with transgenic insertion of code for enhanced green fluorescent protein (EGFP) at the locus for glutamic acid decarboxylase 67 (GAD67), one of two key enzymes for the synthesis of γ-aminobutyric acid (GABA) were used to test whether the morphological properties of these neurons show plasticity with nerve injury. Physiological properties and the delivery of intracellular label to EGFP-expressing lamina II neurons was done using whole-cell patch-clamp in spinal cord slices from sham and chronic constriction injury (CCI) mice. As well, whole cell recordings were made of non-EGFP labeled cells to ascertain changes in overall inhibitory signaling following CCI. The EGFP labeled neurons in both sham and CCI mice exhibited islet, central and vertical cell morphological profiles but no radial cell profiles were observed. The length of cell dendrites was found to be significantly shorter in CCI mice for all cell profile types. The longest neurites averaged 155.96 ± 18.29 μm in CCI mice compared to 334.93 ± 29.48 μm in sham control mice. No change was observed in either passive or evoked membrane properties of EGFP-expressing neurons in CCI versus sham mice. Meanwhile, the frequency of miniature inhibitory post-synaptic currents of non-EGFP expressing spinal lamina II neurons was significantly reduced. These results suggest that reduced inhibitory output from GABA neurons occurs with nerve injury in part due to altered cell morphology.

Evolutionally Conserved Function of Kisspeptin Neuronal System Is Nonreproductive Regulation as Revealed by Nonmammalian Study.

The kisspeptin neuronal system, which consists of a neuropeptide kisspeptin and its receptor Gpr54, is considered in mammals a key factor of reproductive regulation, the so-called hypothalamic-pituitary-gonadal (HPG) axis. However, in nonmammalian vertebrates, especially in teleosts, existence of kisspeptin regulation on the HPG axis is still controversial. In this study, we applied multidisciplinary techniques to a teleost fish, medaka, and examined possible kisspeptin regulation on the HPG axis. First, we generated knockout medaka for kisspeptin-related genes and found that they show normal fertility, gonadal maturation, and expression of gonadotropins. Moreover, the firing activity of GnRH1 neurons recorded by the patch clamp technique was not altered by kisspeptin application. Furthermore, in goldfish, in vivo kisspeptin administration did not show any positive effect on HPG axis regulation. However, as kisspeptin genes are completely conserved among vertebrates except birds, we surmised that kisspeptin should have some important nonreproductive functions in vertebrates. Therefore, to discover novel functions of kisspeptin, we generated a gpr54-1:enhanced green fluorescent protein (EGFP) transgenic medaka, whose gpr54-1-expressing cells are specifically labeled by EGFP. Analysis of neuronal projection of gpr54-1:EGFP-expressing neurons showed that these neurons in the ventrolateral preoptic area project to the pituitary and are probably involved in endocrine regulation other than gonadotropin release. Furthermore, combination of deep sequencing, histological, and electrophysiological analyses revealed various novel neural systems that are under control of kisspeptin neurons-that is, those expressing neuropeptide Yb, cholecystokinin, isotocin, vasotocin, and neuropeptide B. Thus, our new strategy to genetically label receptor-expressing neurons gives insights into various kisspeptin-dependent neuronal systems that may be conserved in vertebrates.

Development of an imaging system for in vivo real-time monitoring of neuronal activity in deep brain of free-moving rats.

We have newly developed a system that allows monitoring of the intensity of fluorescent signals from deep brains of rats transgenically modified to express enhanced green fluorescent protein (eGFP) via an optical fiber. One terminal of the optical fiber was connected to a blue semiconductor laser oscillator/green fluorescence detector. The other terminal was inserted into the vicinity of the eGFP-expressing neurons. Since the optical fiber was vulnerable to twisting stresses caused by animal movement, we also developed a cage in which the floor automatically turns, in response to the turning of the rat's head. This relieved the twisting stress on the optical fiber. The system then enabled real-time monitoring of fluorescence in awake and unrestrained rats over many hours. Using this system, we could continuously monitor eGFP-expression in arginine vasopressin-eGFP transgenic rats. Moreover, we observed an increase of eGFP-expression in the paraventricular nucleus under salt-loading conditions. We then performed in vivo imaging of eGFP-expressing GnRH neurons in the hypothalamus, via a bundle consisting of 3000 thin optical fibers. With the combination of the optical fiber bundle connection to the fluorescence microscope, and the special cage system, we were able to capture and retain images of eGFP-expressing neurons from free-moving rats. We believe that our newly developed method for monitoring and imaging eGFP-expression in deep brain neurons will be useful for analysis of neuronal functions in awake and unrestrained animals for long durations.

Optogenetic activation of serotonergic terminals facilitates GABAergic inhibitory input to orexin/hypocretin neurons.

Orexin/hypocretin neurons play a crucial role in the regulation of sleep/wakefulness, primarily in the maintenance of wakefulness. These neurons innervate wide areas of the brain and receive diverse synaptic inputs including those from serotonergic (5-HT) neurons in the raphe nucleus. Previously we showed that pharmacological application of 5-HT directly inhibited orexin neurons via 5-HT1A receptors. However, it was still unclear how 5-HT neurons regulated orexin neurons since 5-HT neurons contain not only 5-HT but also other neurotransmitters. To reveal this, we generated new triple transgenic mice in which orexin neurons express enhanced green fluorescent protein (EGFP) and 5-HT neurons express channelrhodopsin2 (ChR2). Immunohistochemical studies show that nerve endings of ChR2-expressing 5-HT neurons are in close apposition to EGFP-expressing orexin neurons in the lateral hypothalamic area. Using these mice, we could optogenetically activate 5-HT nerve terminals and record postsynaptic effects from orexin neurons. Activation of nerve terminals of 5-HT neurons directly inhibited orexin neurons via the 5HT1A receptor, and also indirectly inhibited orexin neurons by facilitating GABAergic inhibitory inputs without affecting glutamatergic inputs. Increased GABAergic inhibitory inputs in orexin neurons were confirmed by the pharmacological application of 5-HT. These results suggest that orexin neurons are inhibited by 5-HT neurons, primarily via 5-HT, in both direct and indirect manners.

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 20000-30000 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 invitro. 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.

Properties of spinal lamina III GABAergic neurons in naïve and in neuropathic mice

Nerve injury leads to Aβ-fibre-mediated mechanical allodynia that is in part due to an impaired GABAergic inhibition in the spinal cord dorsal horn. The properties and function of GABAergic neurons in spinal cord lamina III, an area where low-threshold mechanosensitive Aβ-fibres terminate are, however, largely unknown. We used transgenic mice, which express enhanced green fluorescent protein (EGFP) under control of the promoter GAD67. The morphology and neurochemical characteristics of GABAergic, EGFP-expressing neurons were characterized. We assessed active and passive membrane properties of spinal lamina III GABAergic neurons in naïve animals and animals with a chronic constriction injury (CCI) of the sciatic nerve. EGFP-expressing neurons in lamina III were predominantly islet cells (47%), whereas non-EGFP-expressing neurons were largely inverted stalked cells (40%). EGFP-expressing neurons accounted for about 25% of GABAergic neurons in lamina III. Forty-four percent co-expressed glycine, 10% neuronal nitric oxide synthase and 3% co-expressed parvalbumin. We found costaining with protein kinase CβII in 42% of EGFP-expressing neurons but no expression of protein kinase Cγ. Membrane properties and excitability of EGFP-and non-EGFP-expressing neurons from naïve and neuropathic animals were indistinguishable. The most frequent firing pattern was tonic firing (naïve: 35%, neuropathic: 37%) followed by gap firing (naïve: 33%, neuropathic: 25%). Delayed, initial burst and single-spike firing patterns made up the remainder in both groups. A change in membrane excitability or discharge pattern of this group of lamina III GABAergic neurons is unlikely the cause for mechanical allodynia in animals with CCI.

Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain

Gene therapy, in its current configuration, is irreversible and does not allow control over transgene expression in case of side effects. Only few regulated vector systems are available, and none of these has reached clinical applicability yet. The mifepristone (Mfp)-regulated Gene Switch (GS) system is characterized by promising features such as being composed of mainly human components and an approved small-molecule drug as an inducer. However, it has not yet been evaluated in adeno-associated virus (AAV) vectors, neither has it been tested for applicability in viral vectors in the central nervous system (CNS). Here, we demonstrate that the GS system can be used successfully in AAV vectors in the brain, and that short-term induced glial cell line-derived neurotrophic factor (GDNF) expression prevented neurodegeneration in a rodent model of Parkinson's disease (PD). We also demonstrate repeated responsiveness to the inducer Mfp and absence of immunological tissue reactions in the rat brain. Human equivalent dosages of Mfp used in this study were lower than those used safely for treatment of psychiatric threats, indicating that the inducer could be safely applied in patients. Our results suggest that the GS system in AAV vectors is well suited for further development towards clinical applicability.

The κ-Opioid Receptor Agonist U-50488 Blocks Ca2+ Channels in a Voltage- and G Protein–Independent Manner in Sensory Neurons

κ-Opioid receptor (κ-OR) activation is known to play a role in analgesia and central sedation. The purpose of the present study was to examine the effect of the κ-OR agonist, U-50488 (an arylacetamide), on Ca channel currents and the signaling proteins involved in acutely isolated rat dorsal root ganglion (DRG) neurons expressing the putative promoter region of the tetrodotoxin-resistant Na channel (NaV 1.8) that is known to be involved in pain transmission. Acutely isolated rat DRG neurons were transfected with cDNA coding for enhanced green fluorescent protein (EGFP), whose expression is driven by the NaV 1.8 promoter region. Thereafter, the whole-cell variant of the patch-clamp technique was used to record Ca channel currents in neurons expressing EGFP. Exposure of EGFP-expressing DRG neurons to U-50488 (0.3-40 μM) led to voltage-independent inhibition of the Ca channel currents. The modulation of the Ca currents did not appear to be mediated by the Gα protein subfamilies: Gαi/o, Gαs, Gαq/11, Gα14, and Gαz. Furthermore, dialysis of the hydrolysis-resistant GDP analog, GDP-β-S (1 mM), did not affect the U-50488-mediated blocking effect, ruling out involvement of other G protein subunits. Finally, U-50488 (20 μM) blocked Ca channels heterologously expressed in HeLa cells that do not express κ-OR. These results suggest that the antinociceptive actions mediated by U-50488 are likely due to both a direct block of Ca channels in sensory neurons as well as G protein modulation of Ca currents via κ-OR-expressing neurons.

Physiological properties of enkephalin-containing neurons in the spinal dorsal horn visualized by expression of green fluorescent protein in BAC transgenic mice

BackgroundEnkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn.ResultsTo develop a new tool for the identification of in vitro enkephalinergic neurons and to analyze enkephalin promoter activity, we generated transgenic mice for a bacterial artificial chromosome (BAC). Enkephalinergic neurons from these mice expressed enhanced green fluorescent protein (eGFP) under the control of the preproenkephalin (PPE) gene (penk1) promoter. eGFP-positive neurons were distributed throughout the gray matter of the spinal cord, and were primarily observed in laminae I-II and V-VII, in a pattern similar to the distribution pattern of enkephalin-containing neurons. Double immunostaining analysis using anti-enkephalin and anti-eGFP antibodies showed that all eGFP-expressing neurons contained enkephalin. Incubation in the presence of forskolin, an activator of adenylate cyclase, increased the number of eGFP-positive neurons. These results indicate that eGFP expression is controlled by the penk1 promoter, which contains cyclic AMP-responsive elements. Sections obtained from sciatic nerve-ligated mice exhibited increased eGFP-positive neurons on the ipsilateral (nerve-ligated side) compared with the contralateral (non-ligated side). These data indicate that PPE expression is affected by peripheral nerve injury. Additionally, single-neuron RT-PCR analysis showed that several eGFP positive-neurons in laminae I-II expressed glutamate decarboxylase 67 mRNA and that some expressed serotonin type 3 receptors.ConclusionsThese results suggest that eGFP-positive neurons in laminae I-II coexpress enkephalin and γ-aminobutyric acid (GABA), and are activated by forskolin and in conditions of nerve injury. The penk1-eGFP BAC transgenic mouse contributes to the further characterization of enkephalinergic neurons in the transmission and modulation of nociceptive information.

Glycine Receptor Channels in Spinal Motoneurons Are Abnormal in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly evolving and fatal adult-onset neurological disease characterized by progressive degeneration of motoneurons. Our previous study showed that glycinergic innervation of spinal motoneurons is deficient in an ALS mouse model expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1). In this study, we have examined, using whole-cell patch-clamp recordings, glycine receptor (GlyR)-mediated currents in spinal motoneurons from these transgenic mice. We developed a dissociated spinal cord culture model using embryonic transgenic mice expressing enhanced green fluorescent protein (eGFP) driven by the Hb9 promoter. Motoneurons were identified as Hb9-eGFP-expressing (Hb9-eGFP(+)) neurons with a characteristic morphology. To examine GlyRs in ALS motoneurons, we bred G93A-SOD1 mice to Hb9-eGFP mice and compared glycine-evoked currents in cultured Hb9-eGFP(+) motoneurons prepared from G93A-SOD1 embryos and from their nontransgenic littermates. Glycine-evoked current density was significantly smaller in the G93A-SOD1 motoneurons compared with control. Furthermore, the averaged current densities of spontaneous glycinergic miniature IPSCs (mIPSCs) were significantly smaller in the G93A-SOD1 motoneurons than in control motoneurons. No significant differences in GABA-induced currents and GABAergic mIPSCs were observed between G93A-SOD1 and control motoneurons. Quantitative single-cell reverse transcription-PCR found lower GlyRα1 subunit mRNA expression in G93A-SOD1 motoneurons, indicating that the reduction of GlyR current may result from the downregulation of GlyR mRNA expression in motoneurons. Immunocytochemistry demonstrated a decrease of surface postsynaptic GlyR on G93A-SOD1 motoneurons. Our study suggests that selective alterations in GlyR function contribute to inhibitory insufficiency in motoneurons early in the disease process of ALS.

Modulation of synaptic transmission from primary afferents to spinal substantia gelatinosa neurons by group III mGluRs in GAD65-EGFP transgenic mice

Group III metabotropic glutamate receptors (mGluRs) are involved in nociceptive transmission in the spinal cord. However, the cellular mechanism underlying the modulation of synaptic transmission from nociceptive primary afferents to dorsal horn neurons by group III mGluRs has yet to be explored. In this study, we used transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the glutamate decarboxylase (GAD) 65 promoter to identify specific subpopulations of GABAergic inhibitory interneurons. By GABA immunolabeling, we confirmed the majority of GAD65-EGFP-expressing neurons were GABAergic. Because GAD65-EGFP-expressing neurons have not been examined in detail before, we first investigated the physiological properties of GAD65-EGFP- and non-EGFP-expressing neurons in substantia gelatinosa (SG) of the spinal dorsal horn. Membrane properties, such as the resting membrane potential, membrane capacitance, action potential threshold, and action potential height, differed significantly between these two groups of neurons. Most EGFP-expressing neurons displayed a tonic firing pattern (73% of recorded neurons) and received monosynaptic Aδ and/or C primary afferent inputs (85% of recorded neurons). In contrast, we observed a delayed firing pattern in 53% of non-EGFP-expressing neurons. After identifying the physiological properties of EGFP-expressing neurons, we tested the effects of group III mGluRs on synaptic transmission pharmacologically. A group III mGluR agonist, L-AP4, attenuated Aδ fiber-evoked synaptic transmission but did not affect C fiber-evoked synaptic transmission to EGFP-expressing neurons. Similar primary afferent-specific inhibition by L-AP4 was also observed in non-EGFP-expressing neurons. Moreover, Aδ fiber-evoked synaptic transmission was suppressed by a selective mGluR7 agonist, AMN082. These results suggest that modulation of the synaptic transmission from primary afferents to SG neurons by group III mGluR agonist is specific to the type of nociceptive primary afferents but not to the type of target neurons.

Efferent Projections of Prokineticin 2 Expressing Neurons in the Mouse Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) in the hypothalamus is the predominant circadian clock in mammals. To function as a pacemaker, the intrinsic timing signal from the SCN must be transmitted to different brain regions. Prokineticin 2 (PK2) is one of the candidate output molecules from the SCN. In this study, we investigated the efferent projections of PK2-expressing neurons in the SCN through a transgenic reporter approach. Using a bacterial artificial chromosome (BAC) transgenic mouse line, in which the enhanced green fluorescence protein (EGFP) reporter gene expression was driven by the PK2 promoter, we were able to obtain an efferent projections map from the EGFP-expressing neurons in the SCN. Our data revealed that EGFP-expressing neurons in the SCN, hence representing some of the PK2-expressing neurons, projected to many known SCN target areas, including the ventral lateral septum, medial preoptic area, subparaventricular zone, paraventricular nucleus, dorsomedial hypothalamic nucleus, lateral hypothalamic area and paraventricular thalamic nucleus. The efferent projections of PK2-expressing neurons supported the role of PK2 as an output molecule of the SCN.

Direct excitation of spinal GABAergic interneurons by noradrenaline

Endogenous pain control is, in part, mediated by descending inhibition of spinal nociception via spinal release of noradrenaline. Antinociception by activation of descending noradrenergic fibres has partially been attributed to the direct inhibition of nociceptive spinal neurons. Here, we tested the alternative hypothesis: the direct excitation of inhibitory spinal interneurons by noradrenaline. Transverse lumbar spinal cord slices were obtained from adult mice expressing enhanced green fluorescent protein (EGFP) in GABAergic neurons under control of the GAD67 promoter. Recordings were made from a total of 113 EGFP-expressing neurons and non-EGFP-expressing neurons in spinal laminae II and III with the perforated patch–clamp technique. In lamina II, where mainly nociceptive afferents terminate, noradrenaline (20μM) depolarised significantly more EGFP-labelled (41%) than non-EGFP-labelled GABAergic neurons (4%). In contrast, noradrenaline hyperpolarised significantly more non-EGFP-labelled (46%) than EGFP-labelled GABAergic neurons (20%). In lamina III, where low threshold afferents terminate, EGFP-labelled neurons were never depolarised but either hyperpolarised (25%) or not affected (75%) by noradrenaline. Depolarisations of EGFP-labelled lamina II neurons were mimicked by the α1-adrenoceptor agonist phenylephrine (10–20μM) and abolished by the α1-adrenoceptor antagonist prazosin (2μM). Hyperpolarisations of EGFP- and non-EGFP-labelled neurons were abolished by the α2-adrenoceptor antagonist yohimbine (2μM). These results show that noradrenaline directly excites inhibitory (GABAergic) lamina II interneurons in addition to its inhibitory effect on (putatively excitatory) interneurons in superficial spinal dorsal horn. Both effects of noradrenaline constitute a synergism in descending inhibition of nociceptive information in the spinal dorsal horn.

Type A GABA-Receptor-Dependent Synaptic Transmission Sculpts Dendritic Arbor Structure inXenopusTadpolesIn Vivo

The emergence of dendritic arbor structure in vivo depends on synaptic inputs. We tested whether inhibitory GABAergic synaptic transmission regulates Xenopus optic tectal cell dendritic arbor development in vivo by expressing a peptide corresponding to an intracellular loop (ICL) of the gamma2 subunit of type A GABA receptors (GABA(A)R), which is required to anchor GABA(A) receptors to the postsynaptic scaffold. Enhanced green fluorescent protein (EGFP)-tagged ICL (EGFP-ICL) was distributed in a punctate pattern at putative inhibitory synapses, identified by vesicular GABA transporter immunoreactive puncta. ICL expression completely blocked GABA(A)R-mediated transmission in 36% of transfected neurons and significantly reduced GABA(A)R-mediated synaptic currents relative to AMPA receptor-mediated synaptic currents in the remaining transfected neurons without altering release probability or neuronal excitability. Further analysis of ICL-expressing neurons with residual GABA(A)R-mediated inputs showed that the capacity of benzodiazepine to enhance GABAergic synaptic responses was reduced in ICL-expressing neurons, indicating that they were likely depleted of gamma2 subunit-containing GABA(A)R. Neurons expressing a mutant form of ICL were comparable to controls. In vivo time-lapse images showed that ICL-expressing neurons have more sparsely branched dendritic arbors, which expand over larger neuropil areas than EGFP-expressing control neurons. Analysis of branch dynamics indicated that ICL expression affected arbor growth by reducing rates of branch addition. Furthermore, we found that decreasing GABAergic synaptic transmission with ICL expression blocked visual experience dependent dendritic arbor structural plasticity. Our findings establish an essential role for inhibitory GABAergic synaptic transmission in the regulation of dendritic structural plasticity in Xenopus in vivo.

A novel method for RNA interference in neurons using enhanced green fluorescent protein (EGFP)-transgenic rats

RNA interference (RNAi) is the simplest way of examining gene function by inhibiting expression. However, due to the low rate of introducing short interfering RNA (siRNA) into neurons, it is difficult to discriminate into which neurons that have been successfully introduced. Here, we used neurons from transgenic rats expressing enhanced green fluorescent protein (EGFP), and we simultaneously applied small interfering RNAs (siRNAs) against EGFP and a targeted gene to the EGFP-expressing neurons. EGFP fluorescence and immunoreactivity of the protein were then assessed by immunofluorescence microscopy. Quantitative analysis of the immunofluorescence confirmed that loss of EGFP closely correlates with loss of the target protein. These results indicate that this method can be used in a wider range of the neuroscientific research, especially in genome-wide studies.

Long‐distance corticocortical GABAergic neurons in the adult monkey white and gray matter

A subgroup of GABAergic neurons has been reported to project over long distances in several species. Here we demonstrate that long-distance cortically projecting nonpyramidal neurons occur in monkeys in both white and gray matter. Nonpyramidal neurons were first identified morphologically. Visualization of Golgi-like details was achieved by retrograde infection from injections of an adenovirus vector, producing enhanced green fluorescent protein (EGFP) under control of a neuron-specific promoter. Injections in areas V1, V4, TEO, and posterior TE resulted in EGFP-expressing nonpyramidal neurons up to 1.5 cm distant from the injections, mainly in the white matter. Some neurons occurred in the gray matter, mainly in layer 3, but also in layers 5 and 6, and, very occasionally, layer 1. As control, we injected cholera toxin subunit B, a standard retrograde tracer, in V4, and observed a similarly wide distribution of neurons in the white matter. Second, the GABAergic identity of EGFP-expressing nonpyramidal neurons was established by colabeling for EGFP and GAD67 in selected tissue sections. Most neurons positive for EGFP and GAD67 were positive for somatostatin (SS; 90%). Of those neurons positive for EGFP and SS, almost all were also positive for neuronal nitric oxide synthase or m2 muscarinic receptor, but only 23% were also positive for calretinin. None were positive for parvalbumin. We conclude that long-distance projecting GABAergic neurons 1) are phylogenetically conserved, although in monkeys most gray matter neurons are in the upper layers, and 2) are heterogeneous in terms of their neurochemistry, location, and potentially function.

Identification of the adrenoceptor subtypes expressed on GABAergic neurons in the anterior hypothalamic area and rostral zona incerta of GAD65-eGFP transgenic mice

GABA is a major neurotransmitter in the hypothalamus. In particular, neurons in the paraventricular nucleus (PVN) of the hypothalamus receive dense GABAergic inputs from peri-PVN regions. The noradrenergic system has been reported as a modulator of GABAergic transmission to the PVN. Previous electrophysiological and morphological studies support the presence of adrenoceptors on GABAergic neurons innervating the PVN. In this study, we identified three adrenoceptors on GABAergic neurons in the peri-PVN region, focusing on the anterior hypothalamic area (AHA) and rostral zona incerta (ZIr). GABAergic neurons were identified using enhanced green fluorescent protein (eGFP), followed by single cell RT-PCR analysis of the GABA synthetic enzymes, glutamic acid decarboxylase (GAD)65 and/or GAD67. Single cell RT-PCR data revealed the expression of alpha(1A)-, alpha(1B)- and alpha(2A)-adrenoceptor mRNA on GABAergic neurons in AHA and ZIr. Additionally, immunohistochemical studies showed that the immunoreactivities of alpha(1A)-, alpha(1B)- and alpha(2A)-adrenoceptor were colocalized with eGFP-expressing neurons in AHA and ZIr. The present findings suggest the contribution of adrenoceptors to the modulation of GABAergic neurons in AHA and ZIr.

Suitability of hCMV for viral gene expression in the brain

To the editor: Viral gene delivery is a powerful tool that is rapidly gaining popularity in experimental neuroscience. I believe that the near future will see rapid expansion of this remarkably versatile and adaptable method. Recent communication by Wickersham et al.1 describes yet another interesting implementation of this technology for retrograde tracing of neurons from their target areas. The key point of the paper is demonstration of a high level of enhanced GFP (EGFP) expression using a rabies virus mutant with glycoprotein gene substituted by EGFP. After injecting the vector into the thalamus, the authors showed many bright EGFPexpressing cortical pyramidal neurons. As a reference, they used a lentivirus with rabies virus envelope glycoprotein in which EGFP expression was driven by human cytomegalovirus (hCMV) promoter and found a very low level of EGFP expression with it. I would like to draw the attention of Nature Methods’ readers to the issue of suitability of hCMV-driven constructs for targeting brain neurons. According to the experience of our laboratory, hCMV expression in brain is only stable in some neuronal phenotypes, for example, cholinergic neurons such as those found in the nucleus of the hypoglossal nerve or vagal motor neurons. Other neurons, for example, in the adjacent nucleus of the solitary tract (which are glutamate, GABA and noradrenergic neurons) yield extremely poor expression with hCMV-containing adenoand lentiviruses2. Hence, the assumption that hCMV-driven viral constructs are guaranteed to express in all neurons is incorrect. Such constructs are, however, very active in glia, sometimes creating a confusing impression of high level of expression (see Supplementary Fig. 1 online). A striking example of a lack of hCMV activity in medullary neurons was recently given3. Therefore, for the comparison to be fair, it is critical that stable and high-level EGFP expression in pyramidal neurons be demonstrated using the same lentivirus (presumably this would require using viruses coated with vesicular stomatitis glycoprotein rather than rabies virus glycoprotein). Incidentally, we also had demonstrated bright unstained EGFP-expressing noradrenergic neurons targeted using conventional adenoviral vectors injected into spinal cord2. Such cells are easily identifiable in acute slices prepared from pretransfected animals and can be studied using patch clamp. Curiously, it seems that the phenotype of neurons affects their suitability for retrograde transduction, as we never succeeded in achieving the same with glutamatergic neurons, such as cortical pyramidals, which were the target in the study by Wickersham et al.1. Finally, it is likely that the expression profile of hCMV-containing vectors based on adeno-associated virus may obey different rules owing to the effects of the viral inverted terminal repeats4.

The neural EGF family member CALEB/NGC mediates dendritic tree and spine complexity

The development of dendritic arborizations and spines is essential for neuronal information processing, and abnormal dendritic structures and/or alterations in spine morphology are consistent features of neurons in patients with mental retardation. We identify the neural EGF family member CALEB/NGC as a critical mediator of dendritic tree complexity and spine formation. Overexpression of CALEB/NGC enhances dendritic branching and increases the complexity of dendritic spines and filopodia. Genetic and functional inactivation of CALEB/NGC impairs dendritic arborization and spine formation. Genetic manipulations of individual neurons in an otherwise unaffected microenvironment in the intact mouse cortex by in utero electroporation confirm these results. The EGF-like domain of CALEB/NGC drives both dendritic branching and spine morphogenesis. The phosphatidylinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway and protein kinase C (PKC) are important for CALEB/NGC-induced stimulation of dendritic branching. In contrast, CALEB/NGC-induced spine morphogenesis is independent of PI3K but depends on PKC. Thus, our findings reveal a novel switch of specificity in signaling leading to neuronal process differentiation in consecutive developmental events.