Targeting genes to specific neuronal or glial cell types is valuable both for understanding and for repairing brain circuits. Adeno-associated viral vectors (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is a challenge. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that ~11% of these AAVs specifically target expression to neuronal and glial cell types in the mouse retina, mouse brain, non-human primate retina in vivo, and in the human retina in vitro. We demonstrate applications for recording, stimulation, and molecular characterization, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast, and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.Despite the central importance for both basic and translational research, most current technologies available for cell-type-targeting rely on transgenic animals, which limits their applicability. Either the genetic tool that senses or modulates brain function, or the enzyme, such as Cre recombinase, that allows the genetic tool to be conditionally expressed, is expressed from the animal's genome. The inclusion of a transgenic component in the cell-type-targeting strategy excludes its use in therapy for humans, limits its range of application in pre-clinical, non-human primate research, and complicates its use in model organisms such as mice. The development of transgenic non-human primates and mice is costly and slow, especially since cell-type targeting is often applied in the context of other genetic manipulations, such as double or triple gene knockouts, or when targeting different cell types with different tools.Viral vectors for cell-type-targeting may overcome such limitations. AAVs are the most frequently used vectors in both basic research and gene therapy, as they are safe for use in all tested species, including humans and non-human primates, and their production is simple, cheap, and fast (Planul and Dalkara, 2017). They have three important components: the capsid for cell entry, the promoter that drives transgene expression, and the gene of interest to be expressed in the transduced cells, and they drive expression episomally (Duan et al., 1998; Penaud-Budloo et al., 2008). Futhermore, many genetic tools are small enough to fit into AAVs, different AAVs can be injected together, and synthetic AAV capsids allow brain-wide delivery (Deverman et al., 2016).Cell-type-targeting by AAVs could be achieved by engineering the capsid and/or by using specific promoters. Capsid protein mutations can be used to tune the efficacy of

Degenerative changes of photoreceptors and pigment epithelium shown here prior to apoptotic loss of photoreceptors may contribute to functional alterations reported in diabetic human patients and different animal models, thus may serve as a potential model for testing the efficacy of neuroprotective agents in diabetes.

Relevant data on the distribution of color cones are summarized, with special emphasis on the marked dorsoventral asymmetries observed in a number of mammalian species. In addition, an overview is given of studies that demonstrate the coexistence of two visual pigments within the same cone cell. The biological significance of these phenomena is discussed in conjunction with comparative immunocytochemical analyses of subprimate retinas. Based on various cone distribution patterns and temporal and spatial visual pigment coexpression, two models of cone photoreceptor differentiation are suggested.

Ca(2+)-buffer proteins (CaBPs) modulate the temporal and spatial characteristics of transient intracellular Ca(2+)-concentration changes in neurons in order to fine-tune the strength and duration of the output signal. CaBPs have been used as neurochemical markers to identify and trace neurons of several brain loci including the mammalian retina. The CaBP content of retinal neurons, however, varies between species and, thus, the results inferred from animal models cannot be utilised directly by clinical ophthalmologists. Moreover, the shortage of well-preserved human samples greatly impedes human retina studies at the cellular and network level. Our purpose has therefore been to examine the distribution of major CaBPs, including calretinin, calbindin-D28, parvalbumin and the recently discovered secretagogin in exceptionally well-preserved human retinal samples. Based on a combination of immunohistochemistry, Neurolucida tracing and Lucifer yellow injections, we have established a database in which the CaBP marker composition can be defined for morphologically identified cell types of the human retina. Hence, we describe the full CaBP make-up for a number of human retinal neurons, including HII horizontal cells, AII amacrine cells, type-1 tyrosine-hydroxylase-expressing amacrine cells and other lesser known neurons. We have also found a number of unidentified cells whose morphology remains to be characterised. We present several examples of the colocalisation of two or three CaBPs with slightly different subcellular distributions in the same cell strongly suggesting a compartment-specific division of labour of Ca(2+)-buffering by CaBPs. Our work thus provides a neurochemical framework for future ophthalmological studies and renders new information concerning the cellular and subcellular distribution of CaBPs for experimental neuroscience.

Connexin36 (Cx36) constituent gap junctions (GJ) throughout the brain connect neurons into functional syncytia. In the retina they underlie the transmission, averaging and correlation of signals prior conveying visual information to the brain. This is the first study that describes retinal bipolar cell (BC) GJs in the human inner retina, whose function is enigmatic even in the examined animal models. Furthermore, a number of unique features (e.g. fovea, trichromacy, midget system) necessitate a reexamination of the animal model results in the human retina. Well-preserved postmortem human samples of this study are allowed to identify Cx36 expressing BCs neurochemically. Results reveal that both rod and cone pathway interneurons display strong Cx36 expression. Rod BC inputs to AII amacrine cells (AC) appear in juxtaposition to AII GJs, thus suggesting a strategic AII cell targeting by rod BCs. Cone BCs serving midget, parasol or koniocellular signaling pathways display a wealth of Cx36 expression to form homologously coupled arrays. In addition, they also establish heterologous GJ contacts to serve an exchange of information between parallel signaling streams. Interestingly, a prominent Cx36 expression was exhibited by midget system BCs that appear to maintain intimate contacts with bistratified BCs serving other pathways. These findings suggest that BC GJs in parallel signaling streams serve both an intra- and inter-pathway exchange of signals in the human retina.

Background Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a two‐stage strategy to induce rapid regeneration of the remnant liver. The technique has been associated with high mortality and morbidity rates. This study aimed to evaluate mitochondrial function, biogenesis and morphology during ALPPS‐induced liver regeneration. Methods Male Wistar rats (n = 100) underwent portal vein ligation (PVL) or ALPPS. The animals were killed at 0 h (without operation), and 24, 48, 72 or 168 h after intervention. Regeneration rate and proliferation index were assessed. Mitochondrial oxygen consumption and adenosine 5′‐triphosphate (ATP) production were measured. Mitochondrial biogenesis was evaluated by protein level measurements of peroxisome proliferator‐activated receptor γ co‐activator (PGC) 1‐α, nuclear respiratory factor (NRF) 1 and 2, and mitochondrial transcription factor α. Mitochondrial morphology was evaluated by electron microscopy. Results Regeneration rate and Ki‐67 index were significantly raised in the ALPPS group compared with the PVL group (regeneration rate at 168 h: mean(s.d.) 291·2(21·4) versus 245·1(13·8) per cent, P < 0·001; Ki‐67 index at 24 h: 86·9(4·6) versus 66·2(4·9) per cent, P < 0·001). In the ALPPS group, mitochondrial function was impaired 48 h after the intervention compared with that in the PVL group (induced ATP production); (complex I: 361·9(72·3) versus 629·7(165·8) nmol per min per mg, P = 0·038; complex II: 517·5(48·8) versus 794·8(170·4) nmol per min per mg, P = 0·044). Markers of mitochondrial biogenesis were significantly lower 48 and 72 h after ALPPS compared with PVL (PGC1‐α at 48 h: 0·61‐fold decrease, P = 0·045; NRF1 at 48 h: 0·48‐fold decrease, P = 0·028). Mitochondrial size decreased significantly after ALPPS (0·26(0·05) versus 0·40(0·07) μm2; P = 0·034). Conclusion Impaired mitochondrial function and biogenesis, along with the rapid energy‐demanding cell proliferation, may cause hepatocyte dysfunction after ALPPS. Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a well known surgical strategy that combines liver partition and portal vein ligation. This method induces immense regeneration in the future liver remnant. The rapid volume increase is of benefit for resectability, but the mortality and morbidity rates of ALPPS are strikingly high. Moreover, lagging functional recovery of the remnant liver has been reported recently.In this translational study, ALPPS caused an overwhelming inflammatory response that interfered with the peroxisome proliferator‐activated receptor γ co‐activator 1‐α‐coordinated, stress‐induced, mitochondrial biogenesis pathway. This resulted in the accumulation of immature and malfunctioning mitochondria in hepatocytes during the early phase of liver regeneration (bioenergetic destabilization).These findings might explain some of the high morbidity if confirmed in patients.

In diabetes, retinal dysfunctions exist prior to clinically detectable vasculopathy, however the pathology behind these functional deficits is still not fully established. Previously, our group published a detailed study on the retinal histopathology of type 1 diabetic (T1D) rat model, where specific alterations were detected. Although the majority of human diabetic patients have type 2 diabetes (T2D), similar studies on T2D models are practically absent. To fill this gap, we examined Zucker Diabetic Fatty (ZDF) rats - a model for T2D - by immunohistochemistry at the age of 32 weeks. Glial reactivity was observed in all diabetic specimens, accompanied by an increase in the number of microglia cells. Prominent outer segment degeneration was detectable with changes in cone opsin expression pattern, without a decrease in the number of labelled elements. The immunoreactivity of AII amacrine cells was markedly decreased and changes were detectable in the number and staining of some other amacrine cell subtypes, while most other cells examined did not show any major alterations. Overall, the retinal histology of ZDF rats shows a surprising similarity to T1D rats indicating that despite the different evolution of the disease, the neuroretinal cells affected are the same in both subtypes of diabetes.

Retinal connexins (Cx) form gap junctions (GJ) in key circuits that transmit average or synchronize signals. Expression of Cx36, -45, -50 and -57 have been described in many species but there is still a disconcerting paucity of information regarding the Cx makeup of human retinal GJs. We used well-preserved human postmortem samples to characterize Cx36 GJ constituent circuits of the outer plexiform layer (OPL). Based on their location, morphometric characteristics and co-localizations with outer retinal neuronal markers, we distinguished four populations of Cx36 plaques in the human OPL. Three of these were comprised of loosely scattered Cx36 plaques; the distalmost population 1 formed cone-to-rod GJs, population 2 in the mid-OPL formed cone-to-cone GJs, whereas the proximalmost population 4 likely connected bipolar cell dendrites. The fourth population (population 3) of Cx36 plaques conglomerated beneath cone pedicles and connected dendritic tips of bipolar cells that shared a common presynaptic cone. Overall, we show that the human outer retina displays a diverse cohort of Cx36 GJ that follows the general mammalian scheme and display a great functional diversity.