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.     

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.     

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.     

Somatic and neuritic spines on tyrosine hydroxylase–immunopositive cells of rat retina

Dopamine‐ and tyrosine hydroxylase–immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse‐related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD‐95, and PSD‐93), that TH cell somata and tapering neurites are also immunopositive for a γ‐aminobutyric acid (GABA) receptor subunit (GABA_AR_α1), and that a synaptic ribbon‐specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707–1730, 2017. © 2016 Wiley Periodicals, Inc.     

Thorny ganglion cells in marmoset retina: Morphological and neurochemical characterization with antibodies against calretinin

In primates, over 17 morphological types of retinal ganglion cell have been distinguished by their dendritic morphology and stratification, but reliable markers for specific ganglion cell populations are still rare. The calcium binding protein calretinin is known to be expressed in the inner nuclear and the ganglion cell layer of marmoset retina, however, the specific cell type(s) expressing calretinin in the ganglion cell layer are yet to be determined. Here, we identified calretinin positive retinal ganglion cells in the common marmoset Callithrix jacchus. Double labeling with the ganglion cell marker RBPMS demonstrated that the large majority (80%) of the calretinin positive cells in the ganglion cell layer are ganglion cells, and 20% are displaced amacrine cells. The calretinin positive ganglion cells made up on average 12% of the total ganglion cell population outside of the foveal region and their proportion increased with eccentricity. Prelabeling with antibodies against calretinin and subsequent intracellular injection with DiI revealed that the large majority of the injected cells (n = 74) were either narrow thorny or broad thorny ganglion cells, 14 cells were displaced amacrine cells. Narrow thorny cells were further distinguished into outer and inner stratifying cells. In addition, weakly labeled cells with a large soma were identified as parasol ganglion cells. Our results show that three types of thorny ganglion cells in marmoset retina can be identified with antibodies against calretinin. Our findings are also consistent with the idea that the proportion of wide‐field ganglion cell types increases in peripheral retina.     

Morphology and connectivity of the small bistratified A8 amacrine cell in the mouse retina

Amacrine cells comprise ～30 morphological types in the mammalian retina. The synaptic connectivity and function of a few γ‐aminobutyric acid (GABA)ergic wide‐field amacrine cells have recently been studied; however, with the exception of the rod pathway‐specific AII amacrine cell, the connectivity of glycinergic small‐field amacrine cells has not been investigated in the mouse retina. Here, we studied the morphology and connectivity pattern of the small‐field A8 amacrine cell. A8 cells in mouse retina are bistratified with lobular processes in the ON sublamina and arboreal dendrites in the OFF sublamina of the inner plexiform layer. The distinct bistratified morphology was first visible at postnatal day 8, reaching the adult shape at P13, around eye opening. The connectivity of A8 cells to bipolar cells and ganglion cells was studied by double and triple immunolabeling experiments by using various cell markers combined with synaptic markers. Our data suggest that A8 amacrine cells receive glutamatergic input from both OFF and ON cone bipolar cells. Furthermore, A8 cells are coupled to ON cone bipolar cells by gap junctions, and provide inhibitory input via glycine receptor (GlyR) subunit α1 to OFF cone bipolar cells and to ON A‐type ganglion cells. Measurements of spontaneous glycinergic postsynaptic currents and GlyR immunolabeling revealed that A8 cells express GlyRs containing the α2 subunit. The results show that the bistratified A8 cell makes very similar synaptic contacts with cone bipolar cells as the rod pathway‐specific AII amacrine cell. However, unlike AII cells, A8 amacrine cells provide glycinergic input to ON A‐type ganglion cells. J. Comp. Neurol. 523:1529–1547, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

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.     

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.     

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.     

Study of retinal neurodegeneration and maculopathy in diabetic Meriones shawi: A particular animal model with human‐like macula

The purpose of this work was to evaluate a potentially useful animal model, Meriones shawi (M.sh)—developing metabolic X syndrome, diabetes and possessing a visual streak similar to human macula—in the study of diabetic retinopathy and diabetic macular edema (DME). Type 2 diabetes (T2D) was induced by high fat diet administration in M.sh. Body weights, blood glucose levels were monitored throughout the study. Diabetic retinal histopathology was evaluated 3 and 7 months after diabetes induction. Retinal thickness was measured, retinal cell types were labeled by immunohistochemistry and the number of stained elements were quantified. Apoptosis was determined with TUNEL assay. T2D induced progressive changes in retinal histology. A significant decrease of retinal thickness and glial reactivity was observed without an increase in apoptosis rate. Photoreceptor outer segment degeneration was evident, with a significant decrease in the number of all cones and M‐cone subtype, but—surprisingly—an increase in S‐cones. Damage of the pigment epithelium was also confirmed. A decrease in the number and labeling intensity of parvalbumin‐ and calretinin‐positive amacrine cells and a loss of ganglion cells was detected. Other cell types showed no evident alterations. No DME‐like condition was noticed even after 7 months. M.sh could be a useful model to study the evolution of diabetic retinal pathology and to identify the role of hypertension and dyslipidemia in the development of the reported alterations. Longer follow up would be needed to evaluate the potential use of the visual streak in modeling human macular diseases.     

Survey of retinal ganglion cell morphology in marmoset

In primate retina, the midget, parasol, and small bistratified cell populations form the large majority of ganglion cells. In addition, there is a variety of low-density wide-field ganglion cell types that are less well characterized. Here we studied retinal ganglion cells in the common marmoset, Callithrix jacchus, using particle-mediated gene transfer. Ganglion cells were transfected with an expression plasmid for the postsynaptic density 95–green fluorescent protein. The retinas were processed with established immunohistochemical markers for bipolar and/or amacrine cells to determine ganglion cell dendritic stratification. In total over 500 ganglion cells were classified based on their dendritic field size, morphology, and stratification in the inner plexiform layer. Over 17 types were distinguished, including midget, parasol, broad thorny, small bistratified, large bistratified, recursive bistratified, recursive monostratified, narrow thorny, smooth monostratified, large sparse, giant sparse (melanopsin) ganglion cells, and a group that may contain several as yet uncharacterized types. Assuming each characterized type forms a hexagonal mosaic, the midget and parasol cells account for over 80% of all ganglion cells in the central retina but only ∼50% of cells in the peripheral (>2 mm) retina. We conclude that the fovea is dominated by midget and parasol cells, but outside the fovea the ganglion cell diversity in marmoset is likely as great as that reported for nonprimate retinas. Taken together, the ganglion cell types in marmoset retina resemble those described previously in macaque retina with respect to morphology, stratification, and change in proportion across the retina.     

Anatomy and spatial organization of Müller glia in mouse retina

Müller glia, the most abundant glia of vertebrate retina, have an elaborate morphology characterized by a vertical stalk that spans the retina and branches in each retinal layer. Müller glia play diverse, critical roles in retinal homeostasis, which are presumably enabled by their complex anatomy. However, much remains unknown, particularly in mouse, about the anatomical arrangement of Müller cells and their arbors, and how these features arise in development. Here we use membrane‐targeted fluorescent proteins to reveal the fine structure of mouse Müller arbors. We find sublayer‐specific arbor specializations within the inner plexiform layer (IPL) that occur consistently at defined laminar locations. We then characterize Müller glia spatial patterning, revealing how individual cells collaborate to form a pan‐retinal network. Müller cells, unlike neurons, are spread across the retina with homogenous density, and their arbor sizes change little with eccentricity. Using Brainbow methods to label neighboring cells in different colors, we find that Müller glia tile retinal space with minimal overlap. The shape of their arbors is irregular but nonrandom, suggesting that local interactions between neighboring cells determine their territories. Finally, we identify a developmental window at postnatal Days 6 to 9 when Müller arbors first colonize the synaptic layers beginning in stereotyped inner plexiform layer sublaminae. Together, our study defines the anatomical arrangement of mouse Müller glia and their network in the radial and tangential planes of the retina, in development and adulthood. The local precision of Müller glia organization suggests that their morphology is sculpted by specific cell to cell interactions with neurons and each other.     

Identification of AⅡ amacrine,displaced amacrine,and bistratified ganglion cell types in human retina with antibodies against calretinin

Antibodies against calretinin are markers for one type of rod pathway interneuron (AⅡ amacrine cell) in the retina of some but not all mammalian species. The AⅡ cells play a crucial role in night‐time (scotopic) vision and have been proposed as a target for optogenetic restoration of vision in retinal disease. In the present study we aimed to characterize the AⅡ cells in human retina. Postmortem human donor eyes were obtained with ethical approval and processed for calretinin immunofluorescence. Calretinin‐positive somas in the inner nuclear and the ganglion cell layer were filled with the lipophilic dye DiI. The large majority (over 80%) of calretinin‐immunoreactive cells is located in the inner nuclear layer, is immunopositive for glycine transporter 1, and shows the typical morphology of AⅡ amacrine cells. In addition, a small proportion of calretinin‐positive cells in the inner nuclear layer and in the ganglion cell layer is glutamic acid decarboxylase‐positive and shows the morphology of widefield amacrine cells (stellate, semilunar, and thorny amacrine cells). About half of the calretinin cells in the ganglion cell layer are bistratified ganglion cells resembling the small bistratified (presumed blue‐ON/yellow‐OFF) and the G17 ganglion cell previously described in primates. We conclude that in human retina, antibodies against calretinin can be used to identify AⅡ amacrine cells in the inner nuclear layer as well as widefield amacrine and small bistratified ganglion cells in the ganglion cell layer. J. Comp. Neurol. 524:39–53, 2016. © 2015 Wiley Periodicals, Inc.     

Melanopsin expressing human retinal ganglion cells: Subtypes,distribution, and intraretinal connectivity

Intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin belong to a heterogenic population of RGCs which regulate the circadian clock, masking behavior, melatonin suppression, the pupillary light reflex, and sleep/wake cycles. The different functions seem to be associated to different subtypes of melanopsin cells. In rodents, subtype classification has associated subtypes to function. In primate and human retina such classification has so far, not been applied. In the present study using antibodies against N‐ and C‐terminal parts of human melanopsin, confocal microscopy and 3D reconstruction of melanopsin immunoreactive (‐ir) RGCs, we applied the criteria used in mouse on human melanopsin‐ir RGCs. We identified M1, displaced M1, M2, and M4 cells. We found two other subtypes of melanopsin‐ir RGCs, which were named “gigantic M1 (GM1)” and “gigantic displaced M1 (GDM1).” Few M3 cells and no M5 subtypes were labeled. Total cell counts from one male and one female retina revealed that the human retina contains 7283 ± 237 melanopsin‐ir (0.63–0.75% of the total number of RGCs). The melanopsin subtypes were unevenly distributed. Most significant was the highest density of M4 cells in the nasal retina. We identified input to the melanopsin‐ir RGCs from AII amacrine cells and directly from rod bipolar cells via ribbon synapses in the innermost ON layer of the inner plexiform layer (IPL) and from dopaminergic amacrine cells and GABAergic processes in the outermost OFF layer of the IPL. The study characterizes a heterogenic population of human melanopsin‐ir RGCs, which most likely are involved in different functions.     

Functions of corazonin and histamine in light entrainment of the circadian pacemaker in the Madeira cockroach,Rhyparobia maderae

The circadian pacemaker of the Madeira cockroach, Rhyparobia (Leucophaea) maderae, is located in the accessory medulla (AME). Ipsi‐ and contralateral histaminergic compound eyes are required for photic entrainment. Light pulses delay locomotor activity rhythm during the early night and advance it during the late night. Thus, different neuronal pathways might relay either light‐dependent delays or advances to the clock. Injections of neuroactive substances combined with running‐wheel assays suggested that GABA, pigment‐dispersing factor, myoinhibitory peptides (MIPs), and orcokinins (ORCs) were part of both entrainment pathways, whereas allatotropin (AT) only delayed locomotor rhythms at the early night. To characterize photic entrainment further, histamine and corazonin were injected. Histamine injections resulted in light‐like phase delays and advances, indicating that the neurotransmitter of the compound eyes participates in both entrainment pathways. Because injections of corazonin only advanced during the late subjective night, it was hypothesized that corazonin is only part of the advance pathway. Multiple‐label immunocytochemistry in combination with neurobiotin backfills demonstrated that a single cell expressed corazonin in the optic lobes that belonged to the group of medial AME interneurons. It colocalized GABA and MIP but not AT or ORC immunoreactivity. Corazonin‐immunoreactive (‐ir) terminals overlapped with projections of putatively light‐sensitive interneurons from the ipsi‐ and contralateral compound eye. Thus, we hypothesize that the corazonin‐ir medial neuron integrates ipsi‐ and contralateral light information as part of the phase‐advancing light entrainment pathway to the circadian clock. J. Comp. Neurol. 525:1250–1272, 2017. © 2016 Wiley Periodicals, Inc.     

Selective plasticity of hippocampal GABAergic interneuron populations following kindling of different brain regions

The vulnerability and plasticity of hippocampal GABAergic interneurons is a topic of broad interest and debate in the field of epilepsy. In this experiment, we used the electrical kindling model of epilepsy to determine whether seizures that originate in different brain regions have differential effects on hippocampal interneuron subpopulations. Long‐Evans rats received 99 electrical stimulations of the hippocampus, amygdala, or caudate nucleus, followed by sacrifice and immunohistochemical or western blot analyses. We analyzed markers of dendritic (somatostatin), perisomatic (parvalbumin), and interneuron‐selective (calretinin) inhibition, as well as an overall indicator (GAD67) of interneuron distribution across all major hippocampal subfields. Our results indicate that kindling produces selective effects on the number and morphology of different functional classes of GABAergic interneurons. In particular, limbic kindling appears to enhance dendritic inhibition, indicated by a greater number of somatostatin‐immunoreactive (‐ir) cells in the CA1 pyramidal layer and robust morphological sprouting in the dentate gyrus. We also found a reduction in the number of interneuron‐selective calretinin‐ir cells in the dentate gyrus of hippocampal‐kindled rats, which suggests a possible reduction of synchronized dendritic inhibition. In contrast, perisomatic inhibition indicated by parvalbumin immunoreactivity appears to be largely resilient to the effects of kindling. Finally, we found a significant induction in the number of GAD67‐cells in caudate‐kindled rats in the dentate gyrus and CA3 hippocampal subfields. Taken together, our results demonstrate that kindling has subfield‐selective effects on the different functional classes of hippocampal GABAergic interneurons. J. Comp. Neurol. 525:389–406, 2017. © 2016 Wiley Periodicals, Inc.     

Postsynaptic gephyrin clustering controls the development of adult‐born granule cells in the olfactory bulb

In adult rodent olfactory bulb, GABAergic signaling regulates migration, differentiation, and synaptic integration of newborn granule cells (GCs), migrating from the subventricular zone. Here we show that these effects depend on the formation of a postsynaptic scaffold organized by gephyrin—the main scaffolding protein of GABAergic synapses, which anchors receptors and signaling molecules to the postsynaptic density—and are regulated by the phosphorylation status of gephyrin. Using lentiviral vectors to selectively transfect adult‐born GCs, we observed that overexpression of the phospho‐deficient gephyrin mutant eGFP‐gephyrin(S270A), which facilitates the formation of supernumerary GABAergic synapses in vitro, favors dendritic branching and the formation of transient GABAergic synapses on spines, identified by the presence of α2‐GABA_ARs. In contrast, overexpression of the dominant‐negative eGFP‐gephyrin(L2B) (a chimera that is enzymatically active but clustering defective), curtailed dendritic growth, spine formation, and long‐term survival of GCs, pointing to the essential role of gephyrin cluster formation for its function. We could exclude any gephyrin overexpression artifacts, as GCs infected with eGFP‐gephyrin were comparable to those infected with eGFP alone. The opposite effects induced by the two gephyrin mutant constructs indicate that the gephyrin scaffold at GABAergic synapses orchestrates signaling cascades acting on the cytoskeleton to regulate neuronal growth and synapse formation. Specifically, gephyrin phosphorylation emerges as a novel mechanism regulating morphological differentiation and long‐term survival of adult‐born olfactory bulb neurons. J. Comp. Neurol. 523:1998–2016, 2015 © 2015 Wiley Periodicals, Inc.