Gad1-promotor-driven GFP expression in non-GABAergic neurons of the nucleus endopiriformis in a transgenic mouse line

Transgenic animals have become a widely used model to identify and study specific cell types in whole organs. Promotor-driven reporter gene labeling of the cells under investigation has promoted experimental efficacy to a large degree. However, rigorous assessment of transgene expression specificity in these animal models is highly recommended to validate cellular identity and to isolate potentially mislabeled cell populations. Here, we report on one such mislabeled neuron population in a widely used transgenic mouse line in which GABAergic somatostatin-expressing interneurons (SOM^pos INs) are labeled by eGFP (so-called GIN mouse, FVB-Tg(GadGFP)45704Swn/J). These neurons represent a subpopulation of all SOM^pos INs. However, we report here on GFP labeling of non-GABAergic neurons in the nucleus endopiriformis of this mouse line.     

Dopamine D1 receptor expression is bipolar cell type‐specific in the mouse retina

In the retina, dopamine is a key molecule for daytime vision. Dopamine is released by retinal dopaminergic amacrine cells and transmits signaling either by conventional synaptic or by volume transmission. By means of volume transmission, dopamine modulates all layers of retinal neurons; however, it is not well understood how dopamine modulates visual signaling pathways in bipolar cells. Here we analyzed Drd1a‐tdTomato BAC transgenic mice and found that the dopamine D1 receptor (D1R) is expressed in retinal bipolar cells in a type‐dependent manner. Strong tdTomato fluorescence was detected in the inner nuclear layer and localized to type 1, 3b, and 4 OFF bipolar cells and type 5‐2, XBC, 6, and 7 ON bipolar cells. In contrast, type 2, 3a, 5‐1, 9, and rod bipolar cells did not express Drd1a‐tdTomato. Other interneurons were also found to express tdTomato including horizontal cells and a subset (25%) of AII amacrine cells. Diverse visual processing pathways, such as color or motion‐coded pathways, are thought to be initiated in retinal bipolar cells. Our results indicate that dopamine sculpts bipolar cell performance in a type‐dependent manner to facilitate daytime vision. J. Comp. Neurol. 524:2059–2079, 2016. © 2015 Wiley Periodicals, Inc.     

Long-range projections from sparse populations of GABAergic neurons in murine subplate

The murine subplate contains some of the earliest generated populations of neurons in the cerebral cortex, which play an important role in the maturation of cortical inhibition. Here we present multiple lines of evidence, that the subplate itself is only very sparsely populated with GABAergic neurons at postnatal day (P)8. We used three different transgenic mouse lines, each of which labels a subset of GABAergic, ganglionic eminence derived neurons. Dlx5/6-eGFP labels the most neurons in cortex (on average 11% of NEUN+ cells across all layers at P8) whereas CGE-derived Lhx6-Cre::Dlx1-Venus^fl cells are the sparsest (2% of NEUN+ cells across all layers at P8). There is significant variability in the layer distribution of labeled interneurons, with Dlx5/6-eGFP and Lhx6-Cre::R26R-YFP being expressed most abundantly in Layer 5, whereas CGE-derived Lhx6-Cre::Dlx1-Venus^fl cells are least abundant in that layer. All three lines label at most 3% of NEUN+ neurons in the subplate, in contrast to L5, in which up to 30% of neurons are GFP+ in Dlx5/6-eGFP. We assessed all three GABAergic populations for expression of the subplate neuron marker connective tissue growth factor (CTGF). CTGF labels up to two-thirds of NEUN+ cells in the subplate, but was never found to colocalize with labeled GABAergic neurons in any of the three transgenic strains. Despite the GABAergic neuronal population in the subplate being sparse, long-distance axonal connection tracing with carbocyanine dyes revealed that some Gad65-GFP+ subplate cells form long-range axonal projections to the internal capsule or callosum.     

Multiple cell types form the VIP amacrine cell population

Amacrine cells are a heterogeneous group of interneurons that form microcircuits with bipolar, amacrine and ganglion cells to process visual information in the inner retina. This study has characterized the morphology, neurochemistry and major cell types of a VIP-ires-Cre amacrine cell population. VIP-tdTomato and -Confetti (Brainbow2.1) mouse lines were generated by crossing a VIP-ires-Cre line with either a Cre-dependent tdTomato or Brainbow2.1 reporter line. Retinal sections and whole-mounts were evaluated by quantitative, immunohistochemical, and intracellular labeling approaches. The majority of tdTomato and Confetti fluorescent cell bodies were in the inner nuclear layer (INL) and a few cell bodies were in the ganglion cell layer (GCL). Fluorescent processes ramified in strata 1, 3, 4, and 5 of the inner plexiform layer (IPL). All tdTomato fluorescent cells expressed syntaxin 1A and GABA-immunoreactivity indicating they were amacrine cells. The average VIP-tdTomato fluorescent cell density in the INL and GCL was 535 and 24 cells/mm², respectively. TdTomato fluorescent cells in the INL and GCL contained VIP-immunoreactivity. The VIP-ires-Cre amacrine cell types were identified in VIP-Brainbow2.1 retinas or by intracellular labeling in VIP-tdTomato retinas. VIP-1 amacrine cells are bistratified, wide-field cells that ramify in strata 1, 4, and 5, VIP-2A and 2B amacrine cells are medium-field cells that mainly ramify in strata 3 and 4, and VIP-3 displaced amacrine cells are medium-field cells that ramify in strata 4 and 5 of the IPL. VIP-ires-Cre amacrine cells form a neuropeptide-expressing cell population with multiple cell types, which are likely to have distinct roles in visual processing.     

DNER and NFIA are expressed by developing and mature AII amacrine cells in the mouse retina

The present study has taken advantage of publicly available cell type specific mRNA expression databases in order to identify potential genes participating in the development of retinal AII amacrine cells. We profile two such genes, Delta/Notch‐like EGF repeat containing (Dner) and nuclear factor I/A (Nfia), that are each heavily expressed in AII amacrine cells in the mature mouse retina, and which conjointly identify this retinal cell population in its entirety when using antibodies to DNER and NFIA. DNER is present on the plasma membrane, while NFIA is confined to the nucleus, consistent with known functions of each of these two proteins. DNER also identifies some other subsets of retinal ganglion and amacrine cell types, along with horizontal cells, while NFIA identifies a subset of bipolar cells as well as Muller glia and astrocytes. During early postnatal development, NFIA labels astrocytes on the day of birth, AII amacrine cells at postnatal (P) day 5, and Muller glia by P10, when horizontal cells also transiently exhibit NFIA immunofluorescence. DNER, by contrast, is present in ganglion and amacrine cells on P1, also labeling the horizontal cells by P10. Developing AII amacrine cells exhibit accumulating DNER labeling at the dendritic stalk, labeling that becomes progressively conspicuous by P10, as it is in maturity. This developmental time course is consistent with a prospective role for each gene in the differentiation of AII amacrine cells.     

Developmental characterization of Zswim5 expression in the progenitor domains and tangential migration pathways of cortical interneurons in the mouse forebrain

GABAergic interneurons play an essential role in modulating cortical networks. The progenitor domains of cortical interneurons are localized in developing ventral forebrain, including the medial ganglionic eminence (MGE), caudal ganglionic eminence (CGE), preoptic area (POA), and preoptic hypothalamic border domain (POH). Here, we characterized the expression pattern of Zswim5, an MGE-enriched gene in the mouse forebrain. At E11.5–E13.5, prominent Zswim5 expression was detected in the subventricular zone (SVZ) of MGE, POA, and POH, but not CGE of ventral telencephalon where progenitors of cortical interneurons resided. At E15.5 and E17.5, Zswim5 expression remained in the MGE/pallidum primordium and ventral germinal zone. Zswim5 mRNA was markedly decreased after birth and was absent in the adult forebrain. Interestingly, the Zswim5 expression pattern resembled the tangential migration pathways of cortical interneurons. Zswim5-positive cells in the MGE appeared to migrate from the MGE through the SVZ of LGE to overlying neocortex. Indeed, Zswim5 was co-localized with Nkx2.1 and Lhx6, markers of progenitors and migratory cortical interneurons. Double labeling showed that Ascl1/Mash1-positive cells co-expressed Zswim5. Zswim5 expressing cells contained none or at most low levels of Ki67 but co-expressed Tuj1 in the SVZ of MGE. These results suggest that Zswim5 is immediately upregulated as progenitors exiting cell cycle become postmitotic. Given that recent studies have elucidated that the cell fate of cortical interneurons is determined shortly after becoming postmitotic, the timing of Zswim5 expression in early postmitotic interneurons suggests a potential role of Zswim5 in regulation of neurogenesis and tangential migration of cortical interneurons.     

Neuron types in the zebrafish optic tectum labeled by an id2b transgene

The larval zebrafish optic tectum has emerged as a prominent model for understanding how neural circuits control visually guided behaviors. Further advances in this area will require tools to monitor and manipulate tectal neurons with cell type specificity. Here, we characterize the morphology and neurotransmitter phenotype of tectal neurons labeled by an id2b:gal4 transgene. Whole-brain imaging of stable transgenic id2b:gal4 larvae revealed labeling in a subset of neurons in optic tectum, cerebellum, and hindbrain. Genetic mosaic labeling of single neurons within the id2b:gal4 expression pattern enabled us to characterize three tectal neuron types with distinct morphologies and connectivities. The first is a neuron type previously identified in the optic tectum of other teleost fish: the tectal pyramidal neuron (PyrN). PyrNs are local interneurons that form two stratified dendritic arbors and one stratified axonal arbor in the tectal neuropil. The second tectal neuron type labeled by the id2b:gal4 transgene is a projection neuron that forms a stratified dendritic arbor in the tectal neuropil and an axon that exits tectum to form a topographic projection to torus longitudinalis (TL). A third neuron type labeled is a projection neuron with a nonstratified dendritic arbor and a descending axonal projection to tegmentum. These findings establish the id2b:gal4 transgenic as a useful tool for future studies aimed at elucidating the functional role of tectum, TL, and tegmentum in visually guided behaviors.     

Development of ON and OFF cholinergic amacrine cells in the human fetal retina

Choline acetyltransferase (ChAT) expressing retinal amacrine cells are present across vertebrates. These interneurons play important roles in the development of retinal projections to the brain and in motion detection, specifically in generating direction-selective responses to moving stimuli. ChAT amacrine cells typically comprise two spatially segregated populations that form circuits in the ‘ON’ or ‘OFF’ synaptic layers of the inner retina. This stereotypic arrangement is also found across the adult human retina, with the notable exception that ChAT expression is evident in the ON but not OFF layer of the fovea, a region specialized for high-acuity vision. We thus investigated whether the human fovea exhibits a developmental path for ON and OFF ChAT cells that is retinal location-specific. Our analysis shows that at each retinal location, human ON and OFF ChAT cells differentiate, form their separate synaptic layers, and establish non-random mosaics at about the same time. However, unlike in the adult fovea, ChAT immunostaining is initially robust in both ON and OFF populations, up until at least mid-gestation. ChAT expression in the OFF layer in the fovea is therefore significantly reduced after mid-gestation. OFF ChAT cells in the human fovea and in the retinal periphery thus follow distinct maturational paths.     

The temporal profile of retinal cell genesis in the marmoset monkey

The New World marmoset monkey (Callithrix jacchus) has a relatively short gestational period compared with other primates but possesses a retina at a similar stage of maturation by birth. Previous studies have highlighted that the complex fovea of the marmoset undergoes a more rapid postnatal development in comparison with the Macaca monkey, reaching a mature stage earlier than these species. In this current study, we examined the prenatal proliferation profile of cells in the entire retina employing the thymidine analogs and also determined their phenotype by double‐label immunocytochemistry using type‐specific markers. Akin to other primate species, we demonstrate a centroperipheral gradient in the emergence of both neurons and Müller glia with cones, ganglion cells, and horizontal cells generated first in the fovea at fetal day (Fd)70–74 and with the last generated at the retinal edge at Fd115. Rods, bipolar cells, amacrine cells, displaced amacrine cells, and Müller glia were generated between Fd76 and Fd135 along the same gradient. Similar to foveal development, marmoset neuronal generation was rapid, only taking 51% of gestation whereas in Macaca this takes 81%. J. Comp. Neurol. 524:1193–1207, 2016. © 2015 Wiley Periodicals, Inc.     

Cell‐specific and developmental expression of lectican‐cleaving proteases in mouse hippocampus and neocortex

Mounting evidence has demonstrated that a specialized extracellular matrix exists in the mammalian brain and that this glycoprotein‐rich matrix contributes to many aspects of brain development and function. The most prominent supramolecular assemblies of these extracellular matrix glycoproteins are perineuronal nets, specialized lattice‐like structures that surround the cell bodies and proximal neurites of select classes of interneurons. Perineuronal nets are composed of lecticans, a family of chondroitin sulfate proteoglycans that includes aggrecan, brevican, neurocan, and versican. These lattice‐like structures emerge late in postnatal brain development, coinciding with the ending of critical periods of brain development. Despite our knowledge of the presence of lecticans in perineuronal nets and their importance in regulating synaptic plasticity, we know little about the development or distribution of the extracellular proteases that are responsible for their cleavage and turnover. A subset of a large family of extracellular proteases (called a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS]) is responsible for endogenously cleaving lecticans. We therefore explored the expression pattern of two aggrecan‐degrading ADAMTS family members, ADAMTS15 and ADAMTS4, in the hippocampus and neocortex. Here, we show that both lectican‐degrading metalloproteases are present in these brain regions and that each exhibits a distinct temporal and spatial expression pattern. Adamts15 mRNA is expressed exclusively by parvalbumin‐expressing interneurons during synaptogenesis, whereas Adamts4 mRNA is exclusively generated by telencephalic oligodendrocytes during myelination. Thus, ADAMTS15 and ADAMTS4 not only exhibit unique cellular expression patterns but their developmental upregulation by these cell types coincides with critical aspects of neural development. J. Comp. Neurol. 523:629–648, 2015. © 2014 Wiley Periodicals, Inc.     

Coexpression of auxiliary subunits KChIP and DPPL in potassium channel Kv4‐positive nociceptors and pain‐modulating spinal interneurons

Subthreshold A‐type K⁺ currents (I_SAs) have been recorded from the somata of nociceptors and spinal lamina II excitatory interneurons, which sense and modulate pain, respectively. Kv4 channels are responsible for the somatodendritic I_SAs. Accumulative evidence suggests that neuronal Kv4 channels are ternary complexes including pore‐forming Kv4 subunits and two types of auxiliary subunits: K⁺ channel‐interacting proteins (KChIPs) and dipeptidyl peptidase‐like proteins (DPPLs). Previous reports have shown Kv4.3 in a subset of nonpeptidergic nociceptors and Kv4.2/Kv4.3 in certain spinal lamina II excitatory interneurons. However, whether and which KChIP and DPPL are coexpressed with Kv4 in these I_SA‐expressing pain‐related neurons is unknown. In this study we mapped the protein distribution of KChIP1, KChIP2, KChIP3, DPP6, and DPP10 in adult rat dorsal root ganglion (DRG) and spinal cord by immunohistochemistry. In the DRG, we found colocalization of KChIP1, KChIP2, and DPP10 in the somatic surface and cytoplasm of Kv4.3(+) nociceptors. KChIP3 appears in most Aβ and Aδ sensory neurons as well as a small population of peptidergic nociceptors, whereas DPP6 is absent in sensory neurons. In the spinal cord, KChIP1 is coexpressed with Kv4.3 in the cell bodies of a subset of lamina II excitatory interneurons, while KChIP1, KChIP2, and DPP6 are colocalized with Kv4.2 and Kv4.3 in their dendrites. Within the dorsal horn, besides KChIP3 in the inner lamina II and lamina III, we detected DPP10 in most projection neurons, which transmit pain signal to brain. The results suggest the existence of Kv4/KChIP/DPPL ternary complexes in I_SA‐expressing nociceptors and pain‐modulating spinal interneurons. J. Comp. Neurol. 524:846–873, 2016. © 2015 Wiley Periodicals, Inc.     

Peripheral and central anatomical organization of cutaneous afferent subtypes in a rat nociceptive intersegmental spinal reflex

Stimulation of rat segmental dorsal cutaneous nerves (DCNs) evokes the nociceptive intersegmental cutaneus trunci muscle (CTM) reflex. The reflex consists of early and late responses, mediated by Aδ and C fibers, respectively, based on required stimulation strengths, and shows segmental differences in terms of amplitude and duration. We have now investigated whether the peripheral or central anatomy of nociceptive afferent subtypes in different DCNs also vary in a segmental manner. The numbers of different axon subtypes, determined by axon diameter, were analyzed across peripheral DCNs from T6 to L1. The central projections of T7 and T13 DCN afferents were traced using DCN injections of cholera toxin subunit B (CTB) for myelinated A fibers and isolectin B4 (IB4) for unmyelinated C fibers and both labels were quantified in the dorsal horns. Peripheral axon subtype numbers did not differ significantly across DCNs. Centrally, IB4⁺, but not CTB⁺, projection areas were different between T7 and T13, consistent with different segmental CTM neurogram responses. At both levels, A fibers projected to deeper layers of the dorsal horn than did C fibers. These termination sites are consistent with both mono‐ and polysynaptic connections between these afferents and the ascending propriospinal interneurons of the reflex. Also analyzed were the spatial distribution, the synaptic termination, and the glutamatergic transporter profiles of DCN A and C fibers and their relationship to calcitonin gene‐related peptide (CGRP) staining in the dorsal horn.     

Genetic access to neurons in the accessory optic system reveals a role for Sema6A in midbrain circuitry mediating motion perception

The accessory optic system (AOS) detects retinal image slip and reports it to the oculomotor system for reflexive image stabilization. Here, we characterize two Cre lines that permit genetic access to AOS circuits responding to vertical motion. The first (Pcdh9-Cre) labels only one of the four subtypes of ON direction-selective retinal ganglion cells (ON-DS RGCs), those preferring ventral retinal motion. Their axons diverge from the optic tract just behind the chiasm and selectively innervate the medial terminal nucleus (MTN) of the AOS. Unlike most RGC subtypes examined, they survive after optic nerve crush. The second Cre-driver line (Pdzk1ip1-Cre) labels postsynaptic neurons in the MTN. These project predominantly to the other major terminal nucleus of the AOS, the nucleus of the optic tract (NOT). We find that the transmembrane protein semaphorin 6A (Sema6A) is required for the formation of axonal projections from the MTN to the NOT, just as it is for the retinal innervation of the MTN. These new tools permit manipulation of specific circuits in the AOS and show that Sema6A is required for establishing AOS connections in multiple locations.     

Selective synaptic connections in the retinal pathway for night vision

The mammalian retina encodes visual information in dim light using rod photoreceptors and a specialized circuit: rods→rod bipolar cells→AII amacrine cell. The AII amacrine cell uses sign-conserving electrical synapses to modulate ON cone bipolar cell terminals and sign-inverting chemical (glycinergic) synapses to modulate OFF cone cell bipolar terminals; these ON and OFF cone bipolar terminals then drive the output neurons, retinal ganglion cells (RGCs), following light increments and decrements, respectively. The AII amacrine cell also makes direct glycinergic synapses with certain RGCs, but it is not well established how many types receive this direct AII input. Here, we investigated functional AII amacrine→RGC synaptic connections in the retina of the guinea pig (Cavia porcellus) by recording inhibitory currents from RGCs in the presence of ionotropic glutamate receptor (iGluR) antagonists. This condition isolates a specific pathway through the AII amacrine cell that does not require iGluRs: cone→ON cone bipolar cell→AII amacrine cell→RGC. These recordings show that AII amacrine cells make direct synapses with OFF Alpha, OFF Delta and a smaller OFF transient RGC type that co-stratifies with OFF Alpha cells. However, AII amacrine cells avoid making synapses with numerous RGC types that co-stratify with the connected RGCs. Selective AII connections ensure that a privileged minority of RGC types receives direct input from the night-vision pathway, independent from OFF bipolar cell activity. Furthermore, these results illustrate the specificity of retinal connections, which cannot be predicted solely by co-stratification of dendrites and axons within the inner plexiform layer.     

The morphological characterization of orientation‐biased displaced large‐field ganglion cells in the central part of goldfish retina

The vertebrate retina has about 30 subtypes of ganglion cells. Each ganglion cell receives synaptic inputs from specific types of bipolar and amacrine cells ramifying at the same depth of the inner plexiform layer (IPL), each of which is thought to process a specific aspect of visual information. Here, we identified one type of displaced ganglion cell in the goldfish retina which had a large and elongated dendritic field. As a population, all of these ganglion cells were oriented in the horizontal axis and perpendicular to the dorsal–ventral axis of the goldfish eye in the central part of retina. This ganglion cell has previously been classified as Type 1.2. However, the circuit elements which synapse with this ganglion cell are not yet characterized. We found that this displaced ganglion cell was directly tracer‐coupled only with homologous ganglion cells at sites containing Cx35/36 puncta. We further illustrated that the processes of dopaminergic neurons often terminated next to intersections between processes of ganglion cells, close to where dopamine D1 receptors were localized. Finally, we showed that Mb1 ON bipolar cells had ribbon synapses in the axonal processes passing through the IPL and made ectopic synapses with this displaced ganglion cell that stratified into stratum 1 of the IPL. These results suggest that the displaced ganglion cell may synapse with both Mb1 cells using ectopic ribbon synapses and OFF cone bipolar cells with regular ribbon synapses in the IPL to function in both scotopic and photopic light conditions.     

Disruption in dopaminergic innervation during photoreceptor degeneration

Dopaminergic amacrine cells (DACs) release dopamine in response to light‐driven synaptic inputs, and are critical to retinal light adaptation. Retinal degeneration (RD) compromises the light responsiveness of the retina and, subsequently, dopamine metabolism is impaired. As RD progresses, retinal neurons exhibit aberrant activity, driven by AII amacrine cells, a primary target of the retinal dopaminergic network. Surprisingly, DACs are an exception to this physiological change; DACs exhibit rhythmic activity in healthy retina, but do not burst in RD. The underlying mechanism of this divergent behavior is not known. It is also unclear whether RD leads to structural changes in DACs, impairing functional regulation of AII amacrine cells. Here we examine the anatomical details of DACs in three mouse models of human RD to determine how changes to the dopaminergic network may underlie physiological changes in RD. By using rd10, rd1, and rd1/C57 mice we were able to dissect the impacts of genetic background and the degenerative process on DAC structure in RD retina. We found that DACs density, soma size, and primary dendrite length are all significantly reduced. Using a novel adeno‐associated virus‐mediated technique to label AII amacrine cells in mouse retina, we observed diminished dopaminergic contacts to AII amacrine cells in RD mice. This was accompanied by changes to the components responsible for dopamine synthesis and release. Together, these data suggest that structural alterations of the retinal dopaminergic network underlie physiological changes during RD. J. Comp. Neurol. 524:1208–1221, 2016. © 2015 Wiley Periodicals, Inc.     

5HTR3A‐driven GFP labels immature olfactory sensory neurons

The ionotropic serotonin receptor, 5‐HT₃, is expressed by many developing neurons within the central nervous system. Since the olfactory epithelium continues to generate new olfactory sensory neurons (OSNs) throughout life, we investigated the possibility that 5‐HT₃ is expressed in the adult epithelium. Using a transgenic mouse in which the promoter for the 5‐HT_3a subunit drives expression of green fluorescent protein (GFP), we assessed the expression of this marker in the olfactory epithelium of adult mice. Both the native 5‐HT_3a mRNA and GFP are expressed within globose basal cells of the olfactory and vomeronasal epithelium in adult mice. Whereas the 5‐HT_3a mRNA disappears relatively quickly after final cell division, the GFP label persists for about 5 days, thereby labeling immature OSNs in both the main olfactory system and vomeronasal organ. The GFP‐labeled cells include both proliferative globose basal cells as well as immature OSNs exhibiting the hallmarks of ongoing differentiation including GAP43, PGP9.5, but the absence of olfactory marker protein. Some of the GFP‐labeled OSNs show characteristics of more mature yet still developing OSNs including the presence of cilia extending from the apical knob and expression of NaV1.5, a component of the transduction cascade. These findings suggest that 5‐HT_3a is indicative of a proliferative or developmental state, regardless of age, and that the 5‐HT_3AGFP mice may prove useful for future studies of neurogenesis in the olfactory epithelium. J. Comp. Neurol. 525:1743–1755, 2017. © 2016 Wiley Periodicals, Inc.