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Jan 11, 2017 - Leukemia inhibitory factor impairs structural and neurochemical development of rat visual cortex in vivo. Maren Engelhardt a,b, Graziella di ...
Molecular and Cellular Neuroscience 79 (2017) 81–92

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Leukemia inhibitory factor impairs structural and neurochemical development of rat visual cortex in vivo Maren Engelhardt a,b, Graziella di Cristo c,d, Jochen Grabert a, Silke Patz a,e, Lamberto Maffei c, Nicoletta Berardi c, Petra Wahle a,⁎ a

Developmental Neurobiology, Faculty of Biology and Biotechnology, Ruhr-University Bochum, Germany Institute of Neuroanatomy, Medical Faculty Mannheim, CBTM, Heidelberg University, Germany Institute of Neuroscience CNR, Pisa, Italy d CHU Sainte-Justine Research Center and Department of Pediatrics, Université de Montréal, Montreal, QC, Canada e Research Unit for Experimental Neurotraumatology, Department of Neurosurgery, Medical University of Graz, Graz, Austria b c

a r t i c l e

i n f o

Article history: Received 26 July 2016 Revised 25 November 2016 Accepted 29 December 2016 Available online 11 January 2017 Keywords: Leukemia inhibitory factor GABA-ergic interneurons Neurotrophin-4 BDNF TrkB receptor GFAP

a b s t r a c t Minipump infusions into visual cortex in vivo at the onset of the critical period have revealed that the proinflammatory cytokine leukemia inhibitory factor (LIF) delays the maturation of thalamocortical projection neurons of the lateral geniculate nucleus, and tecto-thalamic projection neurons of the superior colliculus, and cortical layer IV spiny stellates and layer VI pyramidal neurons. Here, we report that P12–20 LIF infusion inhibits somatic maturation of pyramidal neurons and of all interneuron types in vivo. Likewise, DIV 12–20 LIF treatment in organotypic cultures prevents somatic growth GABA-ergic neurons. Further, while NPY expression is increased in the LIF-infused hemispheres, the expression of parvalbumin mRNA and protein, Kv3.1 mRNA, calbindin D28k protein, and GAD-65 mRNA, but not of GAD-67 mRNA or calretinin protein is substantially reduced. Also, LIF treatment decreases parvalbumin, Kv3.1, Kv3.2 and GAD-65, but not GAD-67 mRNA expression in OTC. Developing cortical neurons are known to depend on neurotrophins. Indeed, LIF alters neurotrophin mRNA expression, and prevents the growth promoting action of neurotophin-4 in GABA-ergic neurons. The results imply that LIF, by altering neurotrophin expression and/or signaling, could counteract neurotrophin-dependent growth and neurochemical differentiation of cortical neurons. © 2017 Elsevier Inc. All rights reserved.

1. Introduction Leukemia inhibitory factor (LIF) is a cytokine of the interleukin-6 family with striking pleiotropic properties at the interface between the immune and the nervous system (reviewed in (Linker et al., 2009; Nicola and Babon, 2015)). LIF is expressed in cortical interneurons and pyramidal cells (Gadient et al., 1998; Lemke et al., 1996). The expression increases in neurons and in astrocytes after brain injury and ischemia (Banner et al., 1997; Sriram et al., 2004; Suzuki et al., 2009) and seizures (Jankowsky and Patterson, 2001). LIF utilizes the JAK-STAT signal transduction pathway via the LIF receptor β (LIFRβ), which is expressed by cortical interneurons and pyramidal cells (Wirth et al., 1998b), and the ubiquitously expressed common gp130 signaling subunit (Turnley and Bartlett, 2000). Besides, IL-6 family cytokines converge on the MAPK and PI3K/Akt pathways utilized by neurotrophic factors (Uren and Turnley, 2014). Vice versa, the neurotrophins converge on STAT3 (Uren and Turnley, 2014), and for instance the cooperation of nerve ⁎ Corresponding author at: Developmental Neurobiology, Department of Zoology and Neurobiology, ND6/72, Ruhr-University Bochum, 44780 Bochum, Germany. E-mail address: [email protected] (P. Wahle).

http://dx.doi.org/10.1016/j.mcn.2016.12.008 1044-7431/© 2017 Elsevier Inc. All rights reserved.

growth factor (NGF) with LIF or ciliary neurotrophic factor is required for sympathetic axonal regeneration (Pellegrino and Habecker, 2013). During perinatal development, LIF promotes the survival of astrocytes and counteracts the proliferative action of epidermal growth factor (Gadient et al., 1998). LIF has pro-inflammatory actions (Kerr and Patterson, 2004), but also activates protective mechanisms in the context of inflammation (reviewed in (Nicola and Babon, 2015; Sugiura et al., 2000; Suzuki et al., 2009). For example, LIF reduces demyelination in an animal model of spinal cord injury and enhances oligodendrocyte survival (Azari et al., 2006; Butzkueven et al., 2002), helps to prevent in vivo and in vitro degeneration of motor neurons in models of amyotrophic lateral sclerosis (Azari et al., 2001; Giess et al., 2000), and is neuroprotective after ischemic damage (Davis et al., 2015; Rowe et al., 2014; Suzuki et al., 2009). LIF is induced by seizures and regulates the GFAP response of astrocytes and the upregulation of the antiepileptic neuropeptide Y (NPY) in neurons (Holmberg and Patterson, 2006). IL-6 family cytokines modulate neurotrophin expression (Otten et al., 2000) and LIF increases NT3 and promotes axonal growth of cortical projection neurons after injury (Blesch et al., 1999). LIF is imported to the brain, and subcutaneous injection of LIF in neonatal rat results in an increased cortical STAT3 phosphorylation, an increased astrocytic GFAP

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expression, and in the juvenile an impaired development of sensorymotor gating and motor behavior (Watanabe et al., 2004). In contrast, mice with a targeted deletion of LIF display increased motor activity in a test for depression-like behavior (Pechnick et al., 2004). An altered expression of IL-6 family cytokines e.g. with infection or inflammation in fetal or early postnatal life is suspected to be related to the emergence of neuropsychiatric disorders later in life (Brown, 2008; Khansari and Sperlagh, 2012). Interestingly, the LIF gene maps to chromosome 22q12, a mental illness susceptibility locus with strong linkage to schizophrenia (Sutherland et al., 1989). LIF indeed alters the development of neocortical interneurons. For example, LIF maintains a high number of NPY mRNA expressing neurons in slice cultures of rat visual cortex (Wirth et al., 1998a,b). NPY neuron numbers decline when endogenous LIF is neutralized with antibodies as well as upon innervation by thalamic afferents in a co-culture system, since the latter also downregulates cortical LIF mRNA expression (Wahle et al., 2000). The reduction of LIF and NPY expression concurs with an increase of expression of the calcium-binding protein parvalbumin (PARV) (Patz et al., 2004), a marker for mature fast-spiking interneurons essential for cognitive functions (Hu et al., 2014). When infused prior to the critical period of visual cortex plasticity between postnatal day (P) 10 and 20, LIF dramatically inhibits the maturation of subcortical visual pathway neurons: in the infused hemisphere, the projection neurons of the lateral geniculate nucleus (LGN) remain smaller, and strikingly, also the tecto-thalamic calbindin-positive neurons from the stratum opticum of the superior colliculus remain smaller (Wahle et al., 2003). Tectal calbindin-positive neurons form synapses with LGN projection neurons and in agreement with classical neurotrophic concepts, suffer in a transsynaptic, retrograde manner, because their target neurons fail to develop properly. Thus, an excess of cortical LIF causes a retrograde shrinkage of direct and indirect afferent neurons (Wahle et al., 2003). Since soma growth of LGN neurons depends on cortical neurotrophin-4 (NT4) (Riddle et al., 1995; Wahle et al., 2003), these results could point to an antagonistic action of LIF and NT4 in the developing visual system. Antagonistic actions have also been reported in dorsal root ganglion neuron, where neurotrophin-mediated survival is attenuated in the presence of LIF (Krieglstein and Unsicker, 1996). Here, we have analyzed the consequences of excess LIF during cortical neuron development in vivo. Results suggest that LIF delays growth and neurochemical differentiation of cortical neurons. 2. Material and methods 2.1. Animals Pigmented Long Evans rats of mixed gender from the in-house breeding facility at the University of Pisa and Ruhr-University Bochum, respectively, were used. All procedures were approved by the Italian Ministry of Health and the State of North Rhine-Westfalia, respectively, and performed according to EU Directive (86.609.EEC and 2010/63/EU). Throughout the study, all animals had access to food and water ad libitum and were housed in a standard 12/12 h light/dark cycle. 2.2. LIF infusions Osmotic minipumps (model 1007D; Alzet, Palo Alto, CA) were implanted in the left hemisphere as previously described (Lodovichi et al., 2000). Under i.p. tribromoethanol anesthesia (300 mg/kg, Avertin®, Sigma), minipumps were connected via PE tubing to a stainless steel 30 gauge cannula implanted 1 mm lateral to lambda into medial area 18 (secondary visual cortex) of the left hemisphere, leaving primary visual area 17 largely intact. Wounds were sutured and treated with antibiotic powder. Animals were allowed to recover and housed individually with a daily check for 7 days. Pumps were filled with LIF (Alomone Labs) at 0.083 μg/μl saline. Infusion was done in 3 animals at a pumping rate of 0.5 μl/h for 7 days. To control for unspecific effects of the infusion

protocol, 2 animals were infused with cytochrome C (cytC, 8.3 μg/μl, Sigma). As shown previously, cytC infusion does not alter activity (Lodovichi et al., 2000), or evoke a misregulation of mRNA encoding interneuronal marker protein (Engelhardt et al., 2007). Therefore, only soma size data are reported here for comparison. Infusion lasted from P12–20; P20 is the start of the critical period for ocular dominance plasticity in rat.

2.3. Immunohistochemistry and in situ hybridization Animals were sacrificed with an overdose of sodium pentobarbital (60 mg/kg, Narcoren®; Merial) and transcardially perfused with 0.9% saline solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were postfixed for 2 h and cryoprotected in 25% buffered sucrose overnight. Right hemispheres were marked by cuts in the ventral cortex. Blocks containing visual cortex, LGN and superior colliculus were frozen in Tissue Tek. Serial, coronal sections of 30 μm thickness were cut with a cryostat. One series of sections was mounted on gelatin-coated slides and stained with thionin. Alternating series of sections were processed for immunohistochemistry and in situ hybridization as previously described (Engelhardt et al., 2007; Wahle et al., 2000; Wirth et al., 1998b). Immunohistochemistry was performed with antibodies against neuropeptide Y (NPY, 1:1000; rabbit; Biotrend, Köln, Germany, Biotrend Cat# BT67-3020-04, RRID:AB_2154010), somatostatin (SOM, 1:500; rabbit; Biotrend, Köln, Germany, Biotrend Cat# BT83-3002-55, RRID:AB_2195933), parvalbumin (PARV, 1:1000; mouse; SWANT, Marly, Switzerland, Swant Cat# 235, RRID:AB_ 10000343), calbindin-D28k (CB, 1:1000; mouse; SWANT, Marly, Switzerland, Swant Cat# 300, RRID:AB_10000347), calretinin (CR, 1:1000; mouse; SWANT, Marly, Switzerland, Swant Cat# 6B3, RRID:AB_ 10000320), the pan-neuronal marker NeuN (1:2000; mouse; Millipore via Merck, Darmstadt, Germany, Millipore Cat# MAB377, RRID:AB_ 2298772), glial fibrillary acidic protein (GFAP, 1:1000; goat; Dako, Hamburg, Germany), myelin basic protein (MBP: 1:1000; Abcam Cat# ab7349, RRID:AB_305869), and developed by the ABC-horseradish peroxidase method (biotinylated secondary antibodies and ABC reagent from Dako, Hamburg, Germany) using diaminobenzidine as chromogen. For in situ hybridization, digoxigenin-UTP (Roche, Mannheim, Germany) labeled antisense riboprobes were synthetized by in vitro transcription from linearized plasmid cDNA encoding NPY, PARV, GAD-67, GAD-65, and Kv3.1. Hybridization and staining using sheep anti-digoxigenin (Roche, Mannheim, Germany) was performed as previously described (Engelhardt et al., 2007; Wahle et al., 2000; Wirth et al., 1998b).

2.4. LIF microinjections Four P25 animals received multiple LIF injections into visual cortex covering areas 17 and 18a. Surgery was performed under i.p. tribromoethanol anesthesia (300 mg/kg, Avertin®, Sigma) as described (Lodovichi et al., 2000). A 2.5 × 2.5 mm region of area 17 and 18a was exposed (2.6–4 mm lateral of the midline and about 2 mm anterior from lambda). LIF (Alomone Labs) was dissolved at 100 ng/ml in 0.9% NaCl. Injections were performed over 50–60 min at 12–16 grid positions spaced 0.5 mm apart with a microinjector (Sutter Instruments, Novato, CA, USA). At every position, 2 × 250 nl were injected at about 700 μm and 300 μm cortical depth. Two control animals received saline injections. One hour (2 LIF injected) and 3 h (2 LIF injected, 2 saline controls) after the injections, animals were sacrificed by an overdose of urethane (2000 mg/kg, i.p.) and transcardially perfused with ice-cold saline. Injected visual cortex and non-injected visual cortex from the contralateral hemisphere and the ipsi- and contralateral parietal cortex directly anterior to the injected field were sampled, frozen on dry ice, and lysates were submitted to PCR analysis.

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2.5. Preparation of organotypic cultures (OTC) Organotypic cortex cultures (OTCs) were prepared as described (Patz et al., 2004; Wahle et al., 2000; Wirth et al., 1998b) from pigmented Long-Evans rats from the in-house breeding facility with approval from the Ruhr-University Animal Research Board and the State of North Rhine-Westfalia. Briefly, visual cortex was explanted at postnatal day 0/P1 (P0, day of birth) and cut into 350 μm thick coronal slices using a McIlwain tissue chopper. Cultures were fed three times a week with semi-artificial medium containing 25% adult horse serum, 25% Hank's balanced Salt Solution, 50% Eagle's Basal Medium, 1 mM L-glutamine (all from Life Technologies, Karlsruhe, Germany) and 0.65% D-Glucose (Merck, Darmstadt, Germany). To inhibit glial growth, 10 μl of an antimitotic cocktail consisting of uridine, cytosine-β-D-arabino-furanosid and 5-fluorodeoxyuridine (each stock 1 mM, all from Sigma) was applied at DIV 2 for 24 h. BDNF, NT4, NT3, NGF or LIF were applied at 20 ng/ml medium once (acute treatment), or long-term every second day from DIV 10–20. OTC were picked from the coverslips, frozen on dry ice, and lysates were submitted to PCR analysis. Generally, all OTC were evaluated carefully to exhibit a maximum in culture integrity with no evident scarring or other blemishes that would indicate any form of deterioration of overall culture quality.

2.6. PCR analysis MRNA was isolated using a Dynabead mRNA Direct Kit (Dynal, Hamburg, Germany), and cDNA libraries were synthesized each with mRNA from 5 OTC with Sensiscript reverse transcriptase (20 U/μl; Qiagen, Hilden, Germany) at 37 °C for 60 min. PCR was performed in a total volume of 50 μl with Taq DNA Polymerase (0.5 U/μL; Qiagen). The mRNAs/ amplicons analyzed here and the PCR conditions were previously published: base pairs 154–393 for BDNF, 309–605 for NT4, 456–946 for NGF, 158–634 for NT3, 179–513 for LIF, 1182–1547 for LIFβR, 1422– 1931 for TrkB kinase domain, and 1459–1943 for TrkC kinase domain (Patz and Wahle, 2006; Patz et al., 2003), 788–1179 for GAD-65, 712– 1312 for GAD-67 (Patz et al., 2003), 137–363 for PARV (Patz et al., 2004), 1395–1866 for KV3.1b, 805–1454 for all splice variants of KV3.2 (Grabert and Wahle, 2009), and 2112–2272 for glucose-6-phosphate dehydrogenase (G6PDH). PCR conditions were kept within the linear range determined for every product. PCR gels were scanned, relative band intensities were determined densitometrically, and normalized to G6PDH expression of the same cDNA library. For the laminar expression analysis, pigmented Long Evans rats aged P0, P5, P12, P25, and P100 were sacrificed with an overdose of sodium pentobarbital (60 mg/kg body weight, Narcoren®, Merial) and transcardially perfused with ice-cold 1× standard sodium citrate buffer. As described previously (Patz and Wahle, 2006), visual cortex was dissected and perpendicular tissue slabs through all layers were frozen on dry ice for PCR analysis (total cortex). Of P5, 12, 25, 100 visual cortex, additional perpendicular tissue slabs were frozen as flat mounts between glass coverslips layers, coronally cryostat-sectioned, slidemounted, dried, fixed with 4% paraformaldehyde, and thionin-stained to determine the vertical dimensions of the cortical layers starting from pial surface using an ocular micrometer scale. Directly adjacent square blocks of 2–3 mm edge length were frozen as flat mounts, mounted with the pial side up, followed by surface-parallel cryostat cutting. Sections containing layers I-III, IV, V and finally VI were collected in RNA extraction buffer, followed by cDNA library synthesis, and PCR for BDNF, NT4 LIF, LIFRβ, and G6PDH for normalization. Only libraries fulfilling the laminar identity test were used: the ether-á-go-go potassium channel is specifically enriched in layer IV, and the PARV expression peaks in layer V as previously described (Patz and Wahle, 2006), and 3 successful preparations were included per age.

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2.7. Cell number and cell size analysis in vivo Following published procedures (Engelhardt et al., 2007; Obst and Wahle, 1997; Patz et al., 2003; Wahle et al., 2003), the number of NPY, GAD-67/65, and PARV mRNA expressing neurons were determined in layers II/III, V and VI in areas 17 and 18a of the ipsilateral (LIF infused) and contralateral hemispheres using a camera lucida at 400× magnification and a rectangular 45,000 μm2 view field ‘sliding’ over the laminar compartments. Neurons within this frame were scored as positive when the staining showed at least a ring of blue reaction product around the nucleus as previously published (Engelhardt et al., 2007; Obst and Wahle, 1997). Next, in alternating sections, the number of NeuN-labeled neurons was determined with the rectangular 45,000 μm2 view field in every laminar compartment at corresponding positions of the ipsi- and the contralateral hemisphere. The proportion of mRNA expressing neurons was then plotted as percentage of NeuN-ir neurons per unit area. For every marker, at least 5 sections were analyzed for every hemisphere. The average percentage in the three laminar compartments in the ipsilateral infused hemisphere was compared to that determined in the contralateral hemisphere. Cross-sectional soma area was determined as previously described (Engelhardt et al., 2007; Wahle et al., 2003). In NeuN-stained sections, all neuronal somatic outlines (nucleus present in the plane of the section) in several randomly chosen view fields in layers II/III and layer V of area 17 were assessed morphometrically at 1000 × magnification with a camera lucida. The soma size of NPY, PARV, SOM, and CR neurons was determined in area 17 starting about 0.3 mm lateral to the infusion site boundary and the corresponding position on the non-infused contralateral hemisphere by sampling all immunoreactive somata in perpendicular stripes from layer I down to the white matter. For every hemisphere, 150 to up to 300 somata were assessed morphometrically from at least 3 sections by operators familiar with this technique, but blinded with respect to the hemisphere analyzed or factor infused. Drawings were digitized and somatic area in μm2 was determined. Ipsilateral (infused hemisphere) and contralateral (untreated hemisphere) values were compared. Biological variability in the two cytC-infused control animals was found to be no larger than ±5% median size shift. 2.8. GABA immunohistochemistry in OTC OTCs were fixed with 4% paraformaldehyde and 0.1 glutaraldehyde in phosphate buffer for 30 min, followed by free-floating fixation for 1.5 h in 4% paraformaldehyde in phosphate buffer (pH 7.4). Subsequently, sections were incubated in 0.5% Triton X-100 in TBS for 1 h and blocked in 3% bovine serum albumin and 3% normal goat serum in TBS. Monoclonal anti-GABA antibody (1: 1000, mouse, Millipore, Billerica, MA, clone 5A9, cat. # MAB316, RRID:AB_94396) was incubated overnight in blocking solution. Biotin-conjugated secondary antibodies were incubated for 3 h at room temperature, followed by 2 h incubation in avidin-biotin-horseradish peroxidase complex (DAKO, Hamburg, Germany). Peroxidase reactivity was developed in 0.02% 3,3′-diaminobenzidine and 0.002% H2O2 in 50 mM Tris buffer for 10 min. Staining was intensified by short incubation with 1% OsO4. Cross-sectional area was determined in drawings of the soma outlines at 1000× with a camera lucida. Per culture, 80–150 somata were sampled in perpendicular stripes from layer I to white matter by operators blinded to the experimental conditions described in Fig. 8.

3. Results 3.1. Developmental expression of LIF and LIF receptor β We analyzed the expression of LIF and LIFRß in developing visual cortex in vivo in comparison to BDNF and NT4 (Fig. 1A–L). BDNF peaked in the fourth postnatal week (Fig. 1A), and the cortical layers express

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Fig. 1. Developmental mRNA expression of BDNF, NT4, LIF, and LIFRβ in rat visual cortex in vivo. (A) BDNF, (B) NT4, (C) LIF, and (D) LIFRβ mRNA expression profiles at five postnatal (P) ages. In A–D, BDNF, NT4, LIF and LIFRβ were amplified from the same cDNA libraries synthetized from mRNA isolated from total cortex blocks at the indicated ages. We analyzed 3 cDNA libraries, each synthetized from one visual cortex at ages indicated, each with 2 × 3 PCR reactions/library. Expression levels are normalized to G6PDH mRNA. Plotted are the library averages as mean ± S.E.M. The average at P0 was set to 1. (E–H) Developmental expression of LIF mRNA in layers I–III, IV, V, and VI. (I–L) Developmental expression of LIFRβ mRNA in the same layers. For E–L, LIF and LIFRβ mRNA were amplified from the same sets of 3 cDNA libraries. They were synthesized from the four laminar compartments isolated from 3 visual cortices (3 animals) per time point. Normalization to G6PDH mRNA was determined in every library. The relative laminar amounts are expressed as percent from total LIF or LIFRβ, resp., mRNA amount determined at the four ages in the total cortex cDNA libraries shown in C and D, respectively. Plotted is the mean ± S.E.M. (M–P) Relative expression of G6PDH mRNA. Levels are similar in the four laminar compartments. Plotted is the mean ± S.E.M. of the densitometric readout obtained in the 3 libraries synthetized per layer and age.

BDNF at equal amounts (Patz and Wahle, 2006). NT4 already peaked during the second and early third postnatal week (Fig. 1B), and the laminar expression shifts with age to become highest in layers I-III (Patz and Wahle, 2006). LIF mRNA in visual cortex steadily declined from birth onwards (Fig. 1C). The laminar profiles revealed that LIF mRNA expression becomes particularly low in thalamo-recipient layer IV (Fig. 1E–H), and this concurs in time with the consolidation of visual thalamic afferent synapses in layer IV. LIFRβ expression peaked at P12 and the profile resembles that of NT4 (Fig. 1D). In the layers, LIFRβ was initially higher in infragranular layers at P5, and about equally expressed in all layers at P12 (Fig. 1I, J). Thereafter, the developmental decline occurred mainly in infragranular layers such that from P25 onwards, LIFRβ was expressed strongest in layers I–III (Fig. 1K, L). In situ hybridization data also reveal a strong expression in supragranular layers (Wirth et al., 1998a). The housekeeping mRNA encoding G6PDH is fairly constantly expressed over age (see Fig. 1 in Patz et al., 2003). Further, it is equally expressed in the layers during development (Fig. 1M–P).

3.2. Neurochemical and morphological deficits after LIF infusion in vivo The infusion of LIF from P12–20 did not alter the histological organization, but the ipsilateral cortex near the infusion site was thinner (Fig. 2A) and neuronal somata appeared smaller (Fig. 2B, C; for quantification see Fig. 4E). GFAP staining of astrocytes was increased with a gradient towards the infusion site (Fig. 2D, E), which was expected because astrocytes activate upon brain tissue lesion, in this case by the infusion canula. Our impression was that astrocyte cell density in the infused hemisphere was not obviously different from the contralateral side. Indeed, LIF directly activates astrocytes without increasing proliferation (Gadient et al., 1998; Suzuki et al., 2009). Myelin basic protein was mainly detected in infragranular layers and in white matter at P20, and resembled the staining seen in the contralateral cortex (Fig. 2F). In situ hybridization revealed a decrease of the density of GAD-65 mRNA expressing neurons (Figs. 3A, B; 4B). By contrast, the density of GAD-67 mRNA expressing neurons was not altered (Figs. 3C; 4A).

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Fig. 2. Histology of LIF-infused cortex. Minipump LIF infusions were made into medial area 18 from P12–20. The depicted regions are from area 17 within 1 mm distance to the infusion site (ipsi, direction marked by asterisk in A) and the homotypic region on the contralateral hemisphere (contra). The ipsi/contra images are arranged such that hemispheres face each other. (A) Thionin staining revealed that the cortex of the infused hemisphere (ipsi) appears thinner. (B) Thionin staining at higher magnification in layer V revealed that cell bodies are smaller in the ipsilateral hemisphere. Arrows mark capillaries in the fields shown in (C). (C) Layer V pyramidal cells in higher magnification. Arrows mark capillaries in the fields shown in (B). (D) GFAP staining was increased in the ipsilateral hemisphere near the infusion site. (E) GFAP staining close to the infusion site at higher magnification revealed intensely labeled astrocytes ipsilaterally. (F) Myelin basic protein staining. Scale bar in A, B = 100 μm, in D, E, F = 150 μm; in C = 20 μm.

Laminar percentages of GAD-67 neurons in ipsi and contralateral cortex (expressed as labeled neurons in percent from NeuN-ir total neurons; Fig. 4A) were at comparable levels. This was similar in cortices infused with the control substance cytC (Engelhardt et al., 2007). It indicated that LIF infusions do not cause a loss of interneurons. In contrast, GAD-65 mRNA was reduced. Percentages of GAD-65 expressing neurons in layers II/III, V and VI were lower throughout area 17 monocular and the laterally positioned binocular segment (Fig. 4B). Labeling intensity of neurons declined towards the infusion site, and close to the infusion site, only a few neurons had remained above detection threshold of the in situ hybridization. Supragranular layers apparently suffered less, but percentages of GAD-65 neurons in layers V and VI were substantially reduced in the monocular segment of area 17, which is closest to the infusion site (Fig. 4B). Percentages of labeled neurons already recovered noticeably in the binocular segment at the lateral border of area 17 (columns labeled “bin” in Fig. 4B). Percentages in area 18a N 5 mm distance to the infusion site were no longer different from contralateral values (Fig. 4B). PARV mRNA (Fig. 3D) and PARV immunoreactivity (Fig. 3E) were almost completely eradicated in area 17 close to the infusion sites. With distance, the staining intensity per neuron gradually recovered, but the percentage of PARV cells was still significantly lower throughout area 18 (Fig. 4C). Further, Kv3.1 mRNA was decreased close to the infusion site and staining intensity recovered with distance (Fig. 3F, G). Kv3.1 is co-expressed in PARV-containing neurons (reviewed in (Hu

et al., 2014), therefore only PARV-expressing cells were quantified. By contrast, NPY mRNA was strongly upregulated and intensely labeled neurons extended up to the infusion site (Fig. 5A, B). The percentage of NPY mRNA expressing neurons in cortical layers was strongly increased in area 17 and through area 18a, although the strength of the effect dissipated with increasing distance to the infusion site (Fig. 4D). In fact, percentages of NPY neurons were similar to the percentages reported for BDNF- and NT4-infused cortex (Engelhardt et al., 2007), and for young organotypic slice cultures (Wahle et al., 2000; Wirth et al., 1998b). Density and staining intensity of intensely calbindin-D28k (CB)-ir bitufted and Martinotti interneurons was not altered (Fig. 5C); these neuron types with columnar dendrites and axons co-express somatostatin (SOM). However, CB was lacking from supragranular pyramidal cells, which usually are weakly CB-ir (Fig. 5C, bracket). Furthermore, staining for calretinin (CR), a marker for bipolar and bitufted cells, was not affected (Fig. 5D). LIF infusion impaired the maturation of neuronal somata, since NeuN-ir pyramidal cells of layers II/III V, PARV-ir, and NPY/SOM-ir somata were significantly smaller in the infused ipsilateral compared to the contralateral hemisphere, and CR-ir cells also tended to be smaller (Fig. 4E). The median size shifts towards negative values shows that in every individual animal, pyramidal cells of layers II/IIII, pyramidal cells of layer V, PARV-ir cells, CR-ir cells, NPY-ir cells, and SOM-ir cells were smaller when close to the LIF infusion site (Fig. 4F). We previously reported that NeuN-ir neurons of layer IV and layer VI neurons were

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Fig. 3. LIF impairs expression of GAD, PARV and Kv3.1. Minipump LIF infusions were made into medial area 18 from P12–20. The pictured regions are from area 17 at the border to or within 1 mm distance to the infusion site (ipsi) and from the homotypic region on the contralateral hemisphere (contra). The ipsi/contra pictures are arranged such that hemispheres face each other. Shown are in situ hybridizations (A) for GAD-65 mRNA, (B) GAD-65 mRNA in supragranular layers at higher magnification, (C) GAD-67 mRNA, (D) PARV mRNA, (E) PARV immunoreactivity (ir), (F) Kv3.1 mRNA expression gradient declining towards the infusion site, (G) Kv3.1 mRNA at higher magnification. Asterisks orient towards or mark the infusion sites; material is taken from different animals (n = 3). Scale bar in A, C, D, E, F = 150 μm, in B, G = 100 μm.

significantly smaller (Wahle et al., 2003), indicating that pyramidal cells and spiny stellates were affected by the LIF infusion. For NPY and PARV neurons, we determined the size variation in area 18a at about 4 mm distance to the infusion site (indexed “far” in Fig. 4F). Indeed, sizes were no longer different from those determined in the homotypic region of the contralateral hemisphere (PARV: median size shift −0.8%; NPY: median size shift −5.0%). CytC-infused control animals did not show significant size shifts (Fig. 4F), which is in line with previous findings (Engelhardt et al., 2007).

3.3. LIF alters the expression of neurotrophins

Infusions of BDNF and NT4, but not NGF, from P12–20 and P20–28 accelerated the expression of interneuron markers and promoted soma size development (Engelhardt et al., 2007). LIF now decreased interneuronal gene expression and soma sizes as shown above and reported earlier (Wahle et al., 2003). Interneurons express TrkB receptors (Gorba and Wahle, 1999) and interneuron development

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Fig. 4. LIF affects interneuronal mRNA expression and soma size maturation. (A) The percentage of GAD-67 mRNA expressing neurons in the layers of visual cortex areas 17 and 18a. (B) The percentage of GAD-65 mRNA expressing neurons. (C) The percentage of PARV mRNA expressing neurons. (D) The percentage of NPY mRNA expressing neurons. In A–D, the laminar percentages of mRNA expressing neurons from total NeuN-ir neurons were determined for layer II/III, V and VI in P12–20 LIF-infused animals (n = 3). Plotted is the mean ± S.E.M. of the averages determined per layer and animal. t-Test ipsi versus contra *p b 0.05. (E) LIF infusion from P12–20 impaired somatic growth. Within 0.5–2 mm lateral to the infusion site, the crosssectional soma area of NeuN-ir pyramidal cells, PARV-ir cells, and peptidergic neurons containing NPY-ir and SOM-ir was lower in the infused hemisphere. Plotted is the mean ± S.E.M. of the averages determined per animal in the contralateral and the infused hemispheres. The number of somata sampled ranged from 87 to 360 per marker and hemisphere (in total, 1939 CR-ir somata, 1114 NPY-ir somata, 3499 PAR-ir somata, 2452 SOM-ir somata, 1743 pyramidal somata of layer II/III, and 1967 pyramidal somata of layer V). t-Test, p values are indicated. (F) Individual variability of the median size shifts in LIF-infused (n = 3) and cytC-infused control animals (n = 2) demonstrates that in every LIF-infused hemisphere, the soma areas of all neuronal subsets were smaller. Median size shifts were computed as follows: (median size ipsi − median size contra) / median size contra; negative values indicate smaller ipsilateral neurons (labeled “i”). Every black dot is the value of one animal. White circles represent median size shifts for these neuronal subsets in cytC-infused control (labeled “c”) animals, these values scattered close to zero (black line); the sizes did not differ between hemispheres as previously reported. PARV-ir and NPY-ir neurons sampled about 4 mm lateral to the infusion site (“far”) had soma areas similar to those in the contralateral hemisphere (median size shifts close to zero), comparable to those seen in cytC-infused animals.

depends on TrkB ligands (Berghuis et al., 2004; Patz et al., 2003, 2004). We hypothesized that LIF might alter the expression of neurotrophins. To test this hypothesis, we applied multiple pico-spritzer injections of LIF into P24 rat visual cortex. Indeed, LIF downregulated NT4 mRNA at 1–3 h post injection (Fig. 6A). Concurrently however, BDNF mRNA increased substantially (Fig. 6A). This was due to the lesion, since BDNF also increased in the saline-injected cortex. Saline injections failed to alter NT4 mRNA expression (Fig. 6A). Moreover, the LIF effect was restricted to the injected region of the visual cortex and did not spread to more rostral parietal cortex (Fig. 6B). The lesion effect of BDNF led us to employ organotypic slice cultures of visual cortex for further analysis of neurotrophin expression after acute and longer-lasting exposure to LIF. About a week after explantation, these cultures are fully regenerated, intrinsically wired like the

cortex and spontaneously active. They allow to analyze direct LIF effects in brain tissue, circumventing any potential influences by the peripheral immune system (Linker et al., 2009), surgery, or anesthesia. We previously reported that LIFRβ is present on pyramidal cells and interneurons (Wirth et al., 1998a). The former express BDNF, NT3, TrkB, and TrkC receptors; the latter can express NT3 and NGF (Biane et al., 2014; Pascual et al., 1998) as well as TrkB receptors (Gorba and Wahle, 1999). The precise cellular source of NT4 in the cortex has not been identified yet, but in vitro, neurons and glial cells can produce NT4 (Otten et al., 2000). First, we tested if LIF mRNA expression in DIV 20 cultures was altered by a 20 ng/ml medium pulse of LIF over the following 1–48 h. This was not the case (Supplementary Fig. S1A). Likewise, LIF mRNA expression was not altered by an acute pulse of NGF, BDNF, NT3, or NT4 (Supplementary Fig. S1B–E). LIFRβ mRNA can be regulated by trophic

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Fig. 5. LIF alters expression of neuropeptide Y. Minipump LIF infusions were made into medial area 18 from P12–20. The depicted regions are from area 17 at the border to or within 1 mm distance of the infusion site (ipsi) and from the homotypic region of the contralateral hemisphere (contra). The ipsi/contra images are arranged such that hemispheres face each other. Shown are (A) in situ hybridizations for NPY mRNA, (B) NPY mRNA in supragranular layers at higher magnification, (C) calbindin-D28K immunoreactivity (CB-ir; brackets demarcate layers II/III), and (D) calretinin immunoreactivity (CR-ir). Asterisks orient towards or mark the infusion sites; material is taken from different animals (n = 3). Scale bar in A, C, D = 150 μm, in B = 100 μm.

factors in neuroblastoma cell lines (Port et al., 2008), but in cortex, LIFRβ mRNA was not altered by BDNF, NT4 or by LIF itself (Supplementary Fig. S1F). Further, in DIV 10–20 long-term stimulated cultures (a total of 5 pulses given every second day), neither LIF, nor BDNF, NT4, NT3 or NGF altered LIF mRNA levels (Supplementary Fig. 1G). This suggested that LIF is neither autoregulated nor regulated by the neurotrophins. Next, we tested whether LIF could regulate the expression of neurotrophin mRNAs. Acute 20 ng/ml medium LIF stimulation in DIV 20 slice cultures immediately decreased NT4 mRNA. It recovered within 12 h and started to exceed control levels by 48 h (Fig. 7A). LIF decreased the mRNA encoding NT3, it recovered and tended to overshoot control levels by 48 h (Fig. 7B). LIF transiently decreased the mRNA encoding NGF, which recovered to control level until 48 h (Fig. 7C). LIF did not alter the mRNA levels of BDNF, and full-length TrkB and TrkC receptors,

which were amplified from the very same sets of cDNA libraries (Fig. 7D–F). Finally, a DIV 10–20 long-term treatment was performed with a total of 5 pulses of LIF given every second day to mimic the in vivo P12–20 infusion period. We found that the expression of TrkC, TrkB, and NGF mRNA was at control levels (Fig. 7G, H). Surprisingly, the level of BDNF mRNA was slightly elevated, and the levels of NT3 and NT4 mRNA were about 4-fold higher (Fig. 7G, H). This suggested that the higher amounts of NT4 observed already 48 h after a single pulse of LIF (Fig. 7A) had continued to ramp up under long-term LIF treatment. Finally, we tested whether the LIF effects observed in vivo could also be evoked in OTC. Expression of interneuronal mRNAs encoding Kv3.1b, Kv3.2, PARV, and GAD-65 mRNA were significantly downregulated in DIV 10–20 LIF-treated OTC. GAD-67 mRNA was not altered (Fig. 8A).

Fig. 6. LIF alters the mRNA expression of NT4. (A) Multiple pico-spritzer injections of LIF (100 ng/ml 0.9% saline, 2 × 250 nl per injection in 300 μm and 700 μm cortical depth at about 20 positions) into area 17/18a followed by survival for 1 h and 3 h. (B) Regional specificity of the LIF injections in the injected visual cortex; PC, parietal cortex anterior to visual cortex. For A and B, per time point 2 animals were injected with LIF, 2 animals were injected with saline to serve as controls. Per animal, one cDNA library was synthetized with mRNA from ipsi- and contralateral visual and parietal cortices, 3 × 3 PCR for BDNF and NT4 per library, normalization to G6PDH; plotted are the averages of the PCR values obtained in every animal as mean ± S.E.M. For statistical testing, the averages from the 1 h and 3 h time points were pooled and tested with the t-test: p b 0.01 for the NT4 downregulation.

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Fig. 7. LIF alters neurotrophin mRNA expression: short-term and long-term kinetic analysis in organotypic cortex cultures. (A–F) Acute effect of one LIF pulse, at DIV 20, on the expression of (A) NT4, (B) NT3, (C) NGF, (D) BDNF, (E) full-length TrkB, and (F) full-length TrkC mRNA expression. For A-F, 3 cDNA libraries were synthetized with mRNA of 5 OTC per condition and time point, 2 × 3 PCR for every mRNA and time point, normalization to G6PDH. The 6 mRNAs were amplified from the same set of libraries. Numbers in bars indicate how many libraries were analyzed. When no change in expression was observed after 2 PCR runs, the procedure was stopped. Plotted are the averages of the PCR values obtained per library as mean ± S.E.M. ANOVA on ranks versus DIV 20 mock stimulated control followed by Holm-Sidak or Dunn's correction (when normality failed) for multiple comparisons identifies a significant downregulation of NT4, NT3 and NGF at the 3 h time point, *p b 0.05. (G, H) Long-term effect of LIF on neurotrophin expression in DIV 20 OTC. LIF stimulation (20 ng/ml medium every second day with the medium exchange from DIV 10) failed to alter the mRNA expression of TrkC, TrkB, and NGF, evoked a small increase of BDNF mRNA, and a 4-fold increase of NT3 and NT4 mRNAs. For G-H, 3 pairs of cDNA libraries (mock stimulated control and factor stimulated) were prepared each from 5 cultures, 2 × 3 PCR per amplicon and library, normalization to G6PDH; the average of mock-stimulated control was set to 1. The graph H has been assembled with TrkC, TrkB, NGF, BDNF, NT3 and NT4 mRNA expression values normalized to mock-stimulated control which is shown only once. Numbers in bars indicate how many libraries were analyzed for every mRNA. Plotted are the averages of the PCR values obtained per library as mean ± S.E.M. t-test *p b 0.05.

Fig. 8. Effects of LIF in organotypic cortex cultures. (A) Expression of interneuronal mRNAs in DIV 10–20 LIF-treated OTC. LIF was applied at 20 ng/ml medium every second day with a medium exchange, control cultures received only fresh medium. Numbers in the bars indicate the number of cDNA libraries, each synthetized from 5 OTC that were analyzed for every mRNA. 4–6 reactions per library and mRNA, normalization to G6PDH; the averages obtained in every library are plotted as mean ± S.E.M. The average of the controls was set to 1, plotted is one control for all. t-test, *p b 0.05. (B) Representative micrographs of GABA-stained somata at DIV 20. Scale bar = 20 μm for all. (C) Soma size analysis of DIV 12 and DIV 20 GABA-ergic interneurons after LIF treatment. Data are normalized to the mean of mock-stimulated controls at DIV 20, which has been set zero. Plotted are the culture averages as mean ± S.E.M. with 80–150 somata per culture sampled in perpendicular stripes from layer I to white matter. Number of cultures analyzed is indicated in the bars. Mann-Whitney U test, p values are given (after correction for multiple testing where appropriate); n.s., not significant.

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This is confirms the in situ hybridization data from our in vivo experiments (Figs. 3, 4). We further analyzed interneuronal growth and since the in vivo study reported that nearly all interneuron subsets are affected, we assessed the cross-sectional areas of GABA-ir somata in OTC (Fig. 8B). DIV 12 cells were naturally smaller than DIV 20 cells (on average 10%). A DIV 8–12 and a DIV 12–20 treatment with NT4 (20 ng/ml medium) increased soma size significantly (Fig. 8C). By contrast, a DIV 12–20 LIF treatment resulted in soma sizes that were on average at the level seen at DIV 12. Strikingly, when NT4 and LIF were coapplied from DIV 12–20, GABA-ergic neurons remained at the average size of DIV 12 neurons and significantly below the average size of mock-stimulated DIV 20 neurons (Fig. 8B, C). This suggested that NT4 fails to promote soma growth in the presence of LIF, and intriguingly this occurred in cultures with strongly upregulated NT3 and NT4 expression (Fig. 7H). Taken together, LIF appears to act upstream of the neurotrophins in visual cortex. 4. Discussion LIF infusions into visual cortex lead to a delay in the maturation of soma sizes of all cortical neuron types and an altered expression of GAD-65, PARV, Kv3.1, and calcium binding proteins. These effects are opposite to those evoked by infusion of TrkB receptor ligands (Engelhardt et al., 2007). Cortical GABAergic interneurons express the TrkB receptor (Gorba and Wahle, 1999) and require TrkB ligands for differentiation (Berghuis et al., 2004; Patz et al., 2003, 2004; Wirth et al., 1998a), whereas pyramidal cells express TrkB and TrkC, and respond to ligands of both receptors. An acute LIF treatment in vivo and in vitro initially decreases NT4, NT3 and NGF mRNA expression, but not BDNF and Trk receptor mRNA expression amplified from the very same set of cDNA libraries. On first glance this might explain the morphological and neurochemical deficits. Unexpectedly however, a long-term LIF treatment leads to a substantially increased NT4 and NT3 mRNA expression, and paradoxically, the deficits persist. During normal development, GAD-65, PARV, Kv3.1, and calbindin in pyramidal cells as well as somatic growth increase between P12–20 (Alcantara et al., 1996; Engelhardt et al., 2007; Grabert and Wahle, 2009; Patz et al., 2003, 2004). As shown here, LIF infusions seem to prevent the increase. The NPY expression decreases with ongoing development, because many neurons express NPY transiently (Obst and Wahle, 1997; Wahle et al., 2000). LIF seems to stabilize NPY expression in neurons which normally express NPY only transiently. Arguments for the specificity of the effect is first, that the expression of some mRNAs and proteins changed while others remain unaltered, second, that the changes occur locally restricted and third, changes occur in a gradient with regard to the infusion site. With increasing distance, they normalize to the staining pattern and the size variation seen in the contralateral cortex. Fourth, cytC or NGF infusions, which produce the same type of lesion, fail to elicit ipsi/contra soma size shifts or differences in marker expression (present study and (Engelhardt et al., 2007). The presence of the normal numerical amounts of GAD-67 mRNA expressing neurons up to the border of the infusion site indicates that long-term LIF infusion does not decrease the proportion of interneurons, at least not during the time window of the present analysis. It has been reported that maternal (prenatal) infection together with perinatal stress causes a loss of PARVcontaining axo-somatic basket and axo-axonic chandelier cells (Giovanoli et al., 2014). Perinatal stress is known to promote cytokine production and a loss of PARV cells has been reported for pro-inflammatory IL-1 and IL-6 when combined with maternal separation stress in adolescents, while the anti-inflammatory IL-10 prevents this loss (Wieck et al., 2013). Likewise, IL-6 causes the death of PARV cells with age in mice (Dugan et al., 2009). Functionally, Kv3.1 and PARV-positive basket and chandelier cells play essential roles for feedforward and feedback inhibition, the generation of up states, and gamma oscillations, a dominant form of synchrony associated with cognition (Hu et al., 2014). During postnatal

development of basket cells, action potential duration and axonal conductance increase, action potential amplitude increases, synaptic release and IPSC kinetics increase, the immature mode of asynchronous GABA release switches to a highly synchronized mode required for gamma oscillations, and this concurs with the developmental upregulation of Kv3 and PARV (Doischer et al., 2008; Jiang et al., 2015; Pangratz-Fuehrer and Hestrin, 2011). Interestingly, in frontal cortex of schizophrenic patients and in animal models for psychiatric diseases, basket and chandelier cells display a reduction of PARV expression and accordingly, a reduction in gamma-oscillations (Cunningham et al., 2006). Cortical NPY-positive neurons express LIFRβ, and LIF activates NPY expression even in electrically silenced neurons (Wirth et al., 1998a). In hippocampus, the glial GFAP expression and the neuronal induction of NPY expression by pilocarpine-evoked seizures requires LIF (Holmberg and Patterson, 2006). High NPY and low PARV expression is indicative of hyperexcitability in animal models for epilepsy and in epilepsy patients; moreover, a lower than normal PARV expression already increases the susceptibility to epilepsy (Schwaller et al., 2004). The high NPY expression observed in the present study could thus be evidence for aberrant activity. NPY is a potent endogenous antiepileptic and neuroprotective peptide (Bacci et al., 2002; Vezzani and Sperk, 2004). It is well established that hyperactivity increases BDNF, NGF, Trk receptor and LIF, but decrease NT3 expression (Jankowsky and Patterson, 2001; Kokaia et al., 1998; Koyama and Ikegaya, 2005). However, this profile differs from the one determined here, showing that LIF and NGF mRNA expression is unchanged, BDNF slightly increased, and NT3, besides NT4, substantially increased under long-term LIF treatment. A LIF-induced increase of NT3 expression has also been reported after cortical lesion (Blesch et al., 1999). Therefore, from a developmental viewpoint, the high NPY expression is interpreted as a symptom of immaturity rather than of hyperactivity. Together, these findings imply that LIF stabilizes immature phenotypes, delays morphological differentiation, and prevents in particular in basket and chandelier cells the timely switch to the adult Kv3.1/PARV-positive phenotype. The acute response of cortical tissue to LIF injections in vivo and a single pulse of LIF in vitro was a reduction of NT3 and NT4 mRNA expression. Only long-term LIF treatment unexpectedly increased the levels of BDNF mRNA slightly, and NT3 and NT4 mRNAs significantly by about 4fold. One might assume that the neurotrophins also remain in expression at more immature levels. For instance, NT3 is highest expressed in fetal cortex, and is strongly expressed at P0 in thalamorecipient layers VI, and at P5 in layer IV because it is essential for visual thalamic afferent ingrowth (Ma et al., 2002; Maisonpierre et al., 1990; Mori et al., 2002; Patz and Wahle, 2006). NT4 peaks between P10 and 20. At this time thalamocortical afferents consolidate the innervation in layer IV. The concurrent decrease of LIF expression in particular in layer IV (Fig. 1) could help to increase the efficacy of NT4 towards thalamocortical fibers, which depend on cortical NT4 (Riddle et al., 1995; Wahle et al., 2000). Indeed, NT4 most powerfully stabilizes deprived-eye inputs during the period of ocular dominance plasticity (Lodovichi et al., 2000). However, the developmental peak of NT4 is notably smaller than the 4-fold increased level observed after long-term LIF treatment. Moreover, BDNF mRNA was not altered by LIF. This rather suggests that the expression of neurotrophins increases with time under LIF treatment. Earlier studies already reported that LIF increases NT3 expression (Blesch et al., 1999), and IL-6 increases in cultured astrocytes within 24 h the expression and release of NGF, NT4 and NT3 protein up to 4fold (Otten et al., 2000). It is intriguing that in the long-term LIF-treated OTC the expression of interneuronal mRNAs as well as the size of the GABA-ergic neurons was substantially decreased despite the high expression of endogenous NT4. A sustained knockdown of Trk ligand expression could have explained the structural and neurochemical immaturity of pyramidal cells and interneurons. However, as mentioned above, the increased expression of BDNF, NT3 and NT4 mRNA after long-term LIF treatment

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stands in conflict with the structural and neurochemical deficits. The interaction of cytokines and neurotrophins are complex and still enigmatic. At this moment, we can only speculate that for instance by disrupting the balance of the JAK-STAT versus the MAPK signaling pathway, excess LIF could attenuate the quality of neurotrophin signaling via the MAPK pathway, which is important for terminal differentiation. For example, by activating gp130 signaling, cytokines can directly upregulate the amount of JAK, STAT3, and gp130 proteins (He et al., 2005; Uren and Turnley, 2014). In olfactory bulb granule cells, LIF-evoked high STAT3 expression levels impair somatodendritic differentiation and promote the maintenance of the progenitor status, whereas low STAT3 expression levels are permissive for terminal differentiation (Yu et al., 2009). Cytokine signaling also upregulates the suppressors of cytokine signaling (SOCS), protein inhibitors of activated STATs, and the SH2 domaincontaining protein-tyrosine phosphatase SHP2, which limit cytokine signaling (Nicola and Babon, 2015; Uren and Turnley, 2014). On one hand, SOCS proteins can interfere in a complex fashion with and even enhance Trk receptor signaling (Uren and Turnley, 2014). On the other hand, by competing with SHP2 for a binding site on the LIFRβ and/or pg130, SOCS proteins could inhibit the LIFRβ-mediated activation of the MAPK pathway (Schmitz et al., 2000). This could impair the cooperativity between cytokines and neurotrophins, possibly by pathway-specific failures which block the growth promoting action of the neurotrophins. Indeed, stimulating LIF-treated cultures with NT4 resulted in a clear-cut failure of NT4 in promoting GABA cell growth. The strong increase of NT4 and NT3 and the small increase of BDNF might then be interpreted as an attempt to strengthen MAPK signaling: cortical cells ‘feel’ they do not receive enough neurotrophic signals and compensatorily upregulate ligand production. Future work has to clarify whether LIF delays terminal differentiation by counteracting neurotrophin signaling in developing neocortex. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mcn.2016.12.008.

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