To begin to unravel the complexities of GABAergic circuits in the superior colliculus (SC), we utilized mouse lines that express green fluorescent protein (GFP) in cells that contain the 67 kDa ...isoform of glutamic acid decarboxylase (GAD67‐GFP), or Cre‐recombinase in cells that contain glutamic acid decarboxylase (GAD; GAD2‐cre). We used Cre‐dependent virus injections in GAD2‐Cre mice and tracer injections in GAD67‐GFP mice, as well as immunocytochemical staining for gamma amino butyric acid (GABA) and parvalbumin (PV) to characterize GABAergic cells that project to the pretectum (PT), ventral lateral geniculate nucleus (vLGN) or parabigeminal nucleus (PBG), and interneurons in the stratum griseum superficiale (SGS) that do not project outside the SC. We found that approximately 30% of SGS neurons in the mouse are GABAergic. Of these GABAergic neurons, we identified three categories of potential interneurons in the GAD67‐GFP line (GABA+GFP ~45%, GABA+GFP + PV ~15%, and GABA+PV ~10%). GABAergic cells that did not contain GFP or PV were identified as potential projection neurons (GABA only ~30%). We found that GABAergic neurons that project to the PBG are primarily located in the SGS and exhibit narrow field vertical, stellate, and horizontal dendritic morphologies, while GABAergic neurons that project to the PT and vLGN are primarily located in layers ventral to the SGS. In addition, we examined GABA and GAD67‐containing elements of the mouse SGS using electron microscopy to further delineate the relationship between GABAergic circuits and retinotectal input. Approximately 30% of retinotectal synaptic targets are the presynaptic dendrites of GABAergic interneurons, and GAD67‐GFP interneurons are a source of these presynaptic dendrites.
We used Cre‐dependent virus injections in GAD2‐Cre mice and tracer injections in GAD67‐ green fluorescent protein (GFP) mice, as well as immunocytochemical staining for gamma amino butyric acid (GABA) and parvalbumin (PV) to characterize GABAergic cells of the stratum griseum superficiale (SGS) via confocal and electron microscopy. The Venn diagram illustrates the approximate proportions of GABAergic SGS neurons listed to the right. GABAergic neurons that contain GFP in the GAD67‐GFP line and/or are labeled with a PV antibody are identified as interneurons. The remaining cells are potential projection neurons that include narrow field vertical, stellate, and horizontal cells that project to the parabigeminal nucleus. The GAD67‐GFP mouse line labels intrinsic interneurons in the SGS, which are a source of GABAergic presynaptic dendrites that receive retinal input and contact nonGABAergic dendrites.
In the dorsal lateral geniculate nucleus (LGN) of mice that lack retinal input, a population of large terminals supplants the synaptic arrangements normally made by the missing retinogeniculate ...terminals. To identify potential sources of these “retinogeniculate replacement terminals,” we used mutant mice (math5–/–) which lack retinofugal projections due to the failure of retinal ganglion cells to develop. In this line, we labeled LGN terminals that originate from the primary visual cortex (V1) or the parabigeminal nucleus (PBG), and compared their ultrastructure to retinogeniculate, V1 or PBG terminals in the dLGN of C57Blk6 (WT) mice (schematically depicted above graph). Corticogeniculate terminals labeled in WT and math5–/– mice were similar in size and both groups were significantly smaller than WT retinogeniculate terminals. In contrast, the PBG projection in math5–/– mice was extensive and there was considerable overlap in the sizes of retinogeniculate terminals in WT mice and PBG terminals in math5–/– mice (summarized in histogram). The data indicate that V1 is not a source of “retinogeniculate replacement terminals” and suggests that large PBG terminals expand their innervation territory to replace retinogeniculate terminals in their absence.
In the dorsal lateral geniculate nucleus (LGN) of mice that lack retinal input, a population of large terminals supplants the synaptic arrangements normally made by the missing retinogeniculate terminals. To identify potential sources of these “retinogeniculate replacement terminals,” we used mutant mice (math5–/–) which lack retinofugal projections due to the failure of retinal ganglion cells to develop. In this line, we labeled LGN terminals that originate from the primary visual cortex (V1) or the parabigeminal nucleus (PBG), and compared their ultrastructure to retinogeniculate, V1, or PBG terminals in the LGN of C57Blk6 (WT) mice (schematically depicted above graph). Corticogeniculate terminals labeled in WT and math5–/– mice were similar in size and both groups were significantly smaller than WT retinogeniculate terminals. In contrast, the PBG projection in math5–/– mice was extensive and there was considerable overlap in the sizes of retinogeniculate terminals in WT mice and PBG terminals in math5–/– mice (summarized in histogram). The data indicate that V1 is not a source of “retinogeniculate replacement terminals” and suggest that large PBG terminals expand their innervation territory to replace retinogeniculate terminals in their absence.
The superior colliculus (SC) is a critical hub for the generation of visually-evoked orienting and defensive behaviors. Among the SC's myriad downstream targets is the parabigeminal nucleus (PBG), ...the mammalian homolog of the nucleus isthmi, which has been implicated in motion processing and the production of defensive behaviors. The inputs to the PBG are thought to arise exclusively from the SC but little is known regarding the precise synaptic relationships linking the SC to the PBG. In the current study, we use optogenetics as well as viral tracing and electron microscopy in mice to better characterize the anatomical and functional properties of the SC-PBG circuit, as well as the morphological and ultrastructural characteristics of neurons residing in the PBG. We characterized GABAergic SC-PBG projections (that do not contain parvalbumin) and glutamatergic SC-PBG projections (which include neurons that contain parvalbumin). These two terminal populations were found to converge on different morphological populations of PBG neurons and elicit opposing postsynaptic effects. Additionally, we identified a population of non-tectal GABAergic terminals in the PBG that partially arise from neurons in the surrounding tegmentum, as well as several organizing principles that divide the nucleus into anatomically distinct regions and preserve a coarse retinotopy inherited from its SC-derived inputs. These studies provide an essential first step toward understanding how PBG circuits contribute to the initiation of behavior in response to visual signals.
Visual pathways of the brain are organized into parallel channels that code different features of the external environment. In the current study, we investigated the anatomical organization of ...parallel pathways from the superior colliculus (SC) to the pulvinar nucleus in the mouse. Virus injections placed in the ipsilateral and contralateral SC to induce the expression of different fluorescent proteins define two pulvinar zones. The lateral pulvinar (Pl) receives ipsilateral SC input and the caudal medial pulvinar (Pcm) receives bilateral SC input. To examine the ultrastructure of these projections using transmission electron microscopy, we injected the SC with viruses to induce peroxidase expression within synaptic vesicles or mitochondria. We quantitatively compared the sizes of ipsilateral and contralateral tectopulvinar terminals and their postsynaptic dendrites, as well as the sizes of the overall population of synaptic terminals and their postsynaptic dendrites in the Pl and Pcm. Our ultrastructural analysis revealed that ipsilateral tectopulvinar terminals are significantly larger than contralateral tectopulvinar terminals. In particular, the ipsilateral tectopulvinar projection includes a subset of large terminals (≥ 1 μm2) that envelop dendritic protrusions of postsynaptic dendrites. We also found that both ipsilateral and contralateral tectopulvinar terminals are significantly larger than the overall population of synaptic terminals in both the Pl and Pcm. Thus, the ipsilateral tectopulvinar projection is structurally distinct from the bilateral tectopulvinar pathway, but both tectopulvinar channels may be considered the primary or “driving” input to the Pl and Pcm.
The confocal image of a coronal section through the left mouse pulvinar nucleus (PUL) illustrates the distribution of projections from the left (Ipsi, magenta) and right (Contra, green) superior colliculus (SC). This study examined the ultrastructure of ipsi and contra SC to PUL projections using viruses that induce the expression of peroxidase in synaptic vesicles (example electron micrographs circled, black arrows indicate synapses). Synaptic terminals originating from the ipsi SC were found to be significantly larger than synaptic terminals originating from the contra SC (left graph), and both populations of SC terminals were found to be significantly larger than synaptic terminals in the PUL that do not originate from the SC (right graph).
A complete picture of signaling pathway to multicellularity is largely unknown. Previously, we generated mutations in a protein prenylation enzyme, GGB, and showed it is essential in maintaining ...multicellularity in the moss Physcomitrium patens (Thole et al. 2014). Here we show that ROP GTPases acts as a downstream factor prenylated by GGB and themselves play an important role in multicellularity of P. patens. We also show that the loss of multicellularity with the suppression of GGB or ROP GTPases is due to uncoordinated cell expansion, defects in cell wall integrity and disturbance of the directional control of cell plate orientation. Expressing prenylatable ROP in the ggb mutant not only rescues multicellularity in protonemata but also results in development of gametophores. While the prenylation of ROP is important for multicellularity, a higher threshold of active ROP is required for gametophore development. Thus our results suggest that ROP activation via the prenylation by GGB is a key process at both the cell and tissue levels, facilitating the developmental transition from one dimension, to two dimensions, and to three dimensions in P. patens.
The following detailed protocol can be applied to demonstrate the localization of GABA receptors in CNS neurons at the ultrastructural level. While others have investigated receptors at the electron ...microscopic level using immunocytochemical techniques, the appearance of the tissue is usually poor and analyses of the distribution of receptors is limited. The methodology described in this paper allows for optimal preservation of the tissue while retaining immunogenicity. It does this, in part, by utilizing a balanced salt solution washout in conjunction with fixation. When the ionic composition of a fixative solution differs from extracellular fluids, like in most fixation protocols for electron microscopy, ultrastructural changes may occur in the tissue. Balanced salt solutions, like the Tyrode solution used here, helps maintain the normal extracellular environment allowing the fixing agent to reach sufficient concentration to bring about permanent and more optimal fixation even when reduced amounts of glutaraldehyde are required to preserve antigenicity. Therefore, unlike many protocols for post-embedding immunoelectronmicroscopy, this method allows for superior preservation of tissue ultrastructure compared to results previously published by others.
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