Mélanie Cavalier

Nadine Crouzin

Azza Ben Sedrine

Marie Celeste

Jesus Ferreira

Janique Guiramand

Catherine Cohen-Solal

Jean-Alain Fehrentz

Jean Martinez

Gérard Barbanel

Dr Michel Vignes
email: michel.vignes@univ-montp2.fr

Involvement of PKA and ERK pathways in ghrelin-induced long-lasting potentiation of excitatory synaptic transmission in the CA1 area of rat hippocampus

Keywords: Hippocampus, plasticity, GHS-R1a, AMPA, NMDA, intracellular calcium

INTRODUCTION

The orexigenic hormone ghrelin, a 28 amino acid octanoylated peptide, was first described as an endogenous peptide predominantly synthetized by the empty stomach, activating hypothalamic neurons responsible for food intake and regulation of energy balance [START_REF] Andrews | Central mechanisms involved in the orexigenic actions of ghrelin[END_REF]. Beside these well-established functions, several reviews mentioned that ghrelin is also associated with the regulation of several brain activities including reward, drug seeking, ethanol addiction, and memory storage (Dickson et al., 2011;[START_REF] Isokawa | Cellular signal mechanisms of reward-related plasticity in the hippocampus[END_REF]. It thus

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provides an interesting link between the central nervous system and the enteric nervous system, so-called 'the second brain'. Moreover, ghrelin exhibits numerous beneficial actions against neurotoxic challenges such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposure [START_REF] Jiang | Ghrelin antagonizes MPTP-induced neurotoxicity to the dopaminergic neurons in mouse substantia nigra[END_REF] or amyloid--1-42 oligomer-induced neurodegeneration [START_REF] Moon | Ghrelin ameliorates cognitive dysfunction and neurodegeneration in intrahippocampal amyloid-beta1-42 oligomer-injected mice[END_REF]. It also prevents amnesia produced by exposure to the N-Methyl-D-Aspartate (NMDA) receptor antagonist dizolcipine (MK801) (Goshadrou et al., 2013).

Ghrelin affects brain activity by binding the growth hormone secretagogue receptor 1a (GHS-R1a), a G protein coupled receptor. GHS-R1a is densely expressed in the hypothalamus, the substantia nigra, the ventral tegmental area (VTA) and throughout the limbic system, including the hippocampus. The activation of GHS-R1a enhances neuronal excitability as demonstrated recently in the substantia nigra pars compacta and CA1 area of the hippocampus [START_REF] Shi | Peptide hormone ghrelin enhances neuronal excitability by inhibition of Kv7/KCNQ channels[END_REF]. This increase in excitability could explain the ghrelininduced increases of dopamine in the nucleus accumbens [START_REF] Jerlhag | Ghrelin stimulates locomotor activity and accumbal dopamine-overflow via central cholinergic systems in mice: implications for its involvement in brain reward[END_REF] and of acetylcholine in the VTA [START_REF] Jerlhag | Concomitant release of ventral tegmental acetylcholine and accumbal dopamine by ghrelin in rats[END_REF].

Furthermore, the contribution of constitutively active GHS-R1a receptors to neurophysiological processes is supported by several studies [START_REF] Pantel | Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature[END_REF][START_REF] Pazos | Basic Aspects of Ghrelin Action[END_REF][START_REF] Portelli | Inactivation of the constitutively active ghrelin receptor attenuates limbic seizure activity in rodents[END_REF]. GHS-R1a is markedly expressed in the hippocampus (Guan et al., 1997), an essential area for learning and memory. Local infusions of ghrelin induce strong pro-mnesic effects as evidenced by spatial memory tests (Diano et al., 2006;[START_REF] Atcha | Cognitive enhancing effects of ghrelin receptor agonists[END_REF][START_REF] Carlini | Ghrelin induced memory facilitation implicates nitric oxide synthase activation and decrease in the threshold to promote LTP in hippocampal dentate gyrus[END_REF][START_REF] Chen | Local infusion of ghrelin enhanced hippocampal synaptic plasticity and spatial memory through activation of phosphoinositide 3-kinase in the dentate gyrus of adult rats[END_REF]. Since long-term potentiation (LTP) is considered as the neurophysiological substrate of memory formation in the CA1 area of the hippocampus [START_REF] Lynch | Long-term potentiation and memory[END_REF], positive effects of ghrelin on synaptic transmission, and most particularly on high frequency-induced LTP, are expected. Consistently, enhancements (Diano et al., 2006;[START_REF] Ribeiro | Ghrelin triggers the synaptic incorporation of AMPA receptors in the hippocampus[END_REF], or facilitation [START_REF] Carlini | Ghrelin induced memory facilitation implicates nitric oxide synthase activation and decrease in the threshold to promote LTP in hippocampal dentate gyrus[END_REF] of LTP elicited by ghrelin are

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This article is protected by copyright. All rights reserved. observed, either using acute hippocampal slice preparations or organotypic cultures. In addition, in vivo experiments in the dentate gyrus show that the application of ghrelin could by itself induce a long-lasting increase in excitatory transmission, while having a positive effect only on the maintenance of high frequency-evoked LTP but not on its induction [START_REF] Chen | Local infusion of ghrelin enhanced hippocampal synaptic plasticity and spatial memory through activation of phosphoinositide 3-kinase in the dentate gyrus of adult rats[END_REF]. These effects of ghrelin likely involve a delayed stimulation of the phosphoinositide-3-kinase (PI3K) and of the extracellular signal-regulated kinases (ERK) pathways [START_REF] Chen | Local infusion of ghrelin enhanced hippocampal synaptic plasticity and spatial memory through activation of phosphoinositide 3-kinase in the dentate gyrus of adult rats[END_REF].

Therefore, we have characterized here the effect of the direct stimulation of GHS-R1a on synaptic transmission in the CA1 area of the hippocampus using selective agonists, i.e. octanoylated-ghrelin and a synthetic analog, JMV1843 (Guerlavais et al., 2003), along to the use of a selective antagonist, JMV2959 [START_REF] Moulin | Toward potent ghrelin receptor ligands based on trisubstituted 1,2,4-triazole structure. 2. Synthesis and pharmacological in vitro and in vivo evaluations[END_REF], in order to characterize these responses. Furthermore, as GHS-R1a could raise neuronal excitability by promoting intracellular Ca 2+ ([Ca 2+ ]i) increases via the coupling to Gq and phospholipase C (PLC) activation, the effect of GHS-R1a agonists was also evaluated on the [Ca 2+ ]i in cultured hippocampal neurons.

MATERIAL AND METHODS

Experiments were conducted on acute rat hippocampal slices and cultured rat hippocampal neurons.

Animals

All experiments were carried out in accordance with the European Community Council Directive of September 22, 2010 (2010/63/UE). This study was approved by the local section of the 'Comité National de Réflexion Ethique sur l'Expérimentation Animale' (C2EA-36).

All efforts were made to minimize animal suffering and to reduce the number of rats used.

Sprague-Dawley rats were from the Centre d'Elevage Depré (France).

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Materials and drugs

Culture media (DMEM/Ham F12 with Hepes and 4.5 g/l glucose), Dulbecco's phosphatebuffered saline (Dulbecco's PBS), Versene™, antibiotics, and fetal calf serum (FCS) were purchased from Invitrogen. Culture dishes were from Nunc.

Ghrelin, D-Amino-phosphonovalerate (AP5) and di-hydroxy-phenyl-glycine (DHPG) were purchased from Abcam Biochemicals. KT-5720, kainate and NMDA were obtained from Sigma and U0126 from Euromedex. KT-5720 and U0126 were used at the efficient concentration recommended by the manufacturer. JMV1843 and JMV2959 were synthesized in the laboratory, and used at efficient doses previously determined [START_REF] Mousseaux | Regulation of ERK1/2 activity by ghrelin-activated growth hormone secretagogue receptor 1A involves a PLC/PKCvarepsilon pathway[END_REF][START_REF] Moulin | The 1,2,4-triazole as a scaffold for the design of ghrelin receptor ligands: development of JMV 2959, a potent antagonist[END_REF].

Hippocampal slice preparation

Hippocampal slices (300 µm) were obtained from male Sprague-Dawley rats. After decapitation, brains were quickly dissected and placed in ice-cold buffer comprising 124 mM NaCl, 3.5 mM KCl, 25 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 1 mM CaCl 2 , 2 mM MgSO 4 , 10 mM D-glucose, and 10 mM HEPES bubbled with O 2 /CO 2 (95%/5%). Slices were then cut with a Vibratome (VT1000S; Leica, France) and maintained at room temperature for at least 1 h in the same buffer supplemented with 1 mM CaCl 2 . This supplemented bufferalso named extracellular mediumwas used for further recordings.

Electrophysiological recordings with micro-electrode array (MEA)

For electrophysiological recordings, slices were transferred to an MEA (MEA60; Multi Channel Systems, Reutlingen, Germany) continually superfused with the above described extracellular medium (flow rate 2 ml.min -1 ) and kept at 32°C. Drugs were directly applied in this superfusion. MEA was positioned on the platform of a Leica inverted microscope

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equipped with a CCD camera (CoolSnap, Roper Scientific, France). Images of the hippocampal slice on the MEA were captured in order to accurately map the synaptic signals recorded in the brain slice. MEA comprised 60 extracellular electrodes [START_REF] Lante | Unveiling novel forms of hippocampal synaptic plasticity with microelectrode arrays[END_REF]:

The inter-electrode distance was 200 μm and each individual electrode from the array could be used either as a recording or as a stimulatory electrode. A nylon mesh was positioned above the slice to obtain a satisfactory electrical contact between the surface of the slice and the electrode array. Stimulation was achieved with an external stimulator (STG-1004; Multi Channel Systems) by applying biphasic current pulses to one electrode of the array, located in the Schaffer Collateral pathway of the hippocampus. Stimulation intensity (50 to 200 μA) and duration (70 to 200 μs) were adapted to avoid multiphasic responses due to excessive stimulation (Heuschkel et al., 2002). Field excitatory postsynaptic potentials (fEPSPs) could then be recorded by all the remaining electrodes of the array at the same time. Signals were recorded and analyzed (MC Rack; Multi Channel Systems). Baseline synaptic signals were evoked using 0.066 Hz frequency stimulation. Slices displaying epileptic-like activity were discarded. Data are presented as means ± SEM on graphs plotting pooled data. They are expressed as fEPSP amplitudes normalized to their corresponding baseline amplitudes. Each individual ('n') corresponds to an experiment performed on a slice from an individual animal.

Primary cultures of rat hippocampal neurons

Primary neuronal cultures were established from 18-day-old embryonic rat hippocampi, as previously described [START_REF] Blanc | Potentiation of glutamatergic agonist-induced inositol phosphate formation by basic fibroblast growth factor is related to developmental features in hippocampal cultures: neuronal survival and glial cell proliferation[END_REF]de Jesus Ferreira et al., 2005), with minor modifications. After pre-incubation with Versene™, hippocampal cells were mechanically dissociated and plated at a density of 2 × 10 6 cells/dish in 8-well dishes containing glass coverslips. Culture dishes and coverslips have been previously coated with poly-L-lysine (15 μg/mL) and then with DMEM/HAM F12 containing 10% FCS. Cells were grown in a

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This article is protected by copyright. All rights reserved. defined medium containing DMEM/HAM F12, supplemented with 33 mM glucose, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 13 mM sodium bicarbonate, 50 μg/mL transferrin, 0.5 μg/mL insulin, 1 pM β-estradiol, 3 nM triiodothyronine, 20 nM progesterone, 46 nM sodium selenite, and 100 μM putrescine. All experiments were performed on cell cultures grown for at least 7 days in vitro.

Measurements of cytosolic-free Ca 2+ concentration

Intracellular calcium concentration ([Ca 2+ ]i) was measured with the fluorescent indicator fura-2 [START_REF] Crouzin | alpha-Tocopherol-mediated long-lasting protection against oxidative damage involves an attenuation of calcium entry through TRP-like channels in cultured hippocampal neurons[END_REF]. For this purpose, hippocampal cells grown on square (10 × 10 mm) glass coverslips were loaded with fura-2 by a 30-min incubation at 37 °C with 5 μM fura-2-AM and 0.02% Pluronic® in the extracellular solution: 124 mM NaCl, 3.5 mM KCl, 25 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 2 mM CaCl 2 , 2 mM MgSO 4 , 10 mM D-Glucose and 10 mM HEPES (bubbled with O 2 /CO 2 : 95/5) pH 7.4. [Ca 2+ ]i was monitored by videomicroscopy. After rinsing, the glass coverslip was transferred to the recording chamber mounted on an inverted microscope (Leica, DMIRB). Fura-2 emission was obtained by exciting alternatively at 340 and 380 nm with a rotating filter wheel (Sutter Instruments) and by monitoring fluorescent emissions (F340 and F380) at 510 nm. The ratio of emissions at 510 nm (F340/F380) was recorded every 2 s. Fluorescent signals were collected with a CCD camera (Hamamatsu), digitized, and analyzed with image analysis software (Acquacosmos, Hamamatsu). Throughout recordings, coverslips were continually superfused with the extracellular solution thermostated at 37 °C. Some experiments were performed while omitting Ca 2+ in the extracellular medium ('Ca 2+ -free' medium). Drug application was performed with a gravity-fed system. The 'n' values represent the entire population of cells recorded in at least 3 independent cultures. On the graphs, data are presented as

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This article is protected by copyright. All rights reserved. averages ± S.E.M. of fura2 fluorescence ratios obtained in individual cells. Ratios have been normalized to their respective baseline value measured prior to any treatment.

Statistics

All statistical analyses were performed using SigmaStat software (SigmaPlot 12.0, Systat Software, San Jose, CA) and the level of significance was settled at P < 0.05. Data are presented as means ± SEM.

One-way analyses of variance followed by Bonferroni method for multiple comparisons were used to test the significance of the level of synaptic potentiation elicited by ghrelin or JMV1843. Two-way analyses of variance followed by Holm-Sidak method for multiple comparisons were used to test the significance of the effect of various drugs on these responses.

For the analyses of calcium transients, areas above peaks in each individual cell were estimated using SigmaPlot software. Values were then compared using either one-way ANOVA on repeated measures for dose-response experiments, or using t-test for comparing two experimental conditions. Finally, the statistical significance of the difference between [Ca 2+ ]i rises elicited by glutamatergic agonists in control and treated cells was evaluated by performing a two-way ANOVA on repeated measured followed by Holm-Sidak method for multiple comparisons.

RESULTS

The application of ghrelin (concentration range: 1-100 nM for 20 min; n = 9 each) elicited an enduring potentiation of the excitatory transmission between Schaffer collaterals and CA1 neurons, which was maintained at least 50 min following ghrelin washout (Figure 1A). The effect of ghrelin was concentration-dependent (F 2,6866 = 441, P < 0.001). It was significantly

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This article is protected by copyright. All rights reserved. enhanced (P < 0.0001) as ghrelin concentration increased from 1 nM to 10 nM in the superfusion medium, but was not significantly different (P = 0.99) when using a further 10-fold higher concentration (Figure 1A). For subsequent experiments, ghrelin was applied at a concentration of 10 nM. Under these conditions, 50 min after ghrelin washout, the fEPSP mean amplitude reached 158 ± 4 % of control amplitude. The paired-pulse facilitation ratios, obtained by applying two pulses separated by 50 ms interval, were 1.22 ± 0.05 (n = 5) prior to ghrelin (10 nM) application and 1.21 ± 0.04 (n = 5) once a stable potentiation had taken place (Figure 1B). These values were not significantly different (P = 0.29), suggesting that the ghrelin-induced synaptic potentiation most likely occurred via postsynaptic mechanisms.

Then we verified that the ghrelin-induced potentiation occurred via the activation of GHS-R1a. For this, the effect of JMV2959, a selective GHS-R1a antagonist, was tested at a concentration which blocked the biological responses elicited by 10 nM ghrelin [START_REF] Moulin | The 1,2,4-triazole as a scaffold for the design of ghrelin receptor ligands: development of JMV 2959, a potent antagonist[END_REF]. In the presence of JMV2959 (100 nM), the effect of ghrelin (10 nM) was completely blocked (F 1,4372 = 1219, P < 0.001 when comparing ghrelin responses either in the absence or the presence of JMV2959; Figure 2A). It is noticeable that JMV2959 had no per se effect on synaptic transmission. Moreover the selective GHS-R1a agonist, JMV1843 (100 nM), also triggered a robust and long-lasting enhancement of synaptic transmission (n = 9, P < 0.001 as compared to basal transmission) (Figure 2B). At the concentration tested, JMV 1843 is known to produce maximal effect on GHS-R1a [START_REF] Mousseaux | Regulation of ERK1/2 activity by ghrelin-activated growth hormone secretagogue receptor 1A involves a PLC/PKCvarepsilon pathway[END_REF].

Fifty min after JMV1843 washout, the fEPSP mean amplitude reached 148 ± 10 % of control amplitude (P < 0.002, as compared to basal). The JMV1843-induced synaptic potentiation was also blocked by JMV2959 (both drugs at 100 nM, n = 5; data not shown).

Next, the dependence on NMDA receptor activation of ghrelin-induced potentiation of fEPSP was tested. To this aim, AP5 (25 µM; n = 9), a selective NMDA receptor antagonist, was applied prior to ghrelin. Under these conditions, the potentiating action of ghrelin was

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significantly enhanced (F 1,4985 = 751, P < 0.001, as compared to the effect of ghrelin in the absence of AP5) and fEPSP mean amplitude reached 240 ± 40 % of control amplitude 50 min after ghrelin wash-out (P = 0.029 as compared to basal; Figure 3). Similar data were obtained when GHS-R1a were activated by JMV1843 (not shown).

The recruitment of intracellular pathways to mediate long-lasting synaptic changes was then further evaluated. In this line, we previously demonstrated that the stimulation of the cAMP/PKA pathway was central to synaptic potentiation and did not require NMDA receptor activation [START_REF] Lante | Developmental switch from LTD to LTP in low frequency-induced plasticity[END_REF]. The application of the selective PKA inhibitor, KT5720 (250 nM), efficiently fully prevented the ghrelin-mediated synaptic potentiation (F 1,3965 = 953, P < 0.001, when comparing the effects of ghrelin either in the absence or the presence of KT5720; Figure 4A). In the presence of KT5720, ghrelin did not elicit any significant modification of synaptic transmission (P = 0.085, as compared to basal transmission). We then verified whether the MAP-kinase pathway, activated after PKA stimulation, could also be involved in ghrelin effect on synaptic transmission. For this, the ERK kinase inhibitor U0126 (10 µM) was tested. This inhibitor also significantly prevented the ghrelin-elicited synaptic potentiation (F 1,4849 = 1082, P < 0.001, when comparing the effects of ghrelin in the absence or the presence of U0126; Figure 4B). Taken together, these data indicate that ghrelin-induced synaptic potentiation results from the activation of GHS-R1a followed by the stimulation of both cAMP/PKA and MAP-kinase pathways.

GHS-R1a may couple the phospholipase C (PLC) pathway thus triggering intracellular

Ca 2+ mobilization after IP3 production [START_REF] Mousseaux | Regulation of ERK1/2 activity by ghrelin-activated growth hormone secretagogue receptor 1A involves a PLC/PKCvarepsilon pathway[END_REF][START_REF] Shi | Peptide hormone ghrelin enhances neuronal excitability by inhibition of Kv7/KCNQ channels[END_REF]. We have thus evaluated the impact of ghrelin and JMV1843 on [Ca 2+ ]i in cultured hippocampal neurons. Both ghrelin (Figure 5A) and JMV1843 (Figure 5B) elicited concentrationdependent increases in [Ca 2+ ]i (P = 0.002 and P < 0.001, respectively). Ghrelin significant effects were detected from a 1 nM concentration (P = 0.008, as compared to basal) and

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reached maximal amplitude at 100 nM. In order to test whether ghrelin-and JMV1843mediated increases in [Ca 2+ ]i effectively resulted from the mobilization of [Ca 2+ ]i from IP3sensitive internal stores, experiments were carried out in Ca 2+ -free extracellular medium. Under these conditions, ghrelin induced changes in [Ca 2+ ]i were strongly reduced (P = 0.0039 and P = 0.002 for the comparison of the effect of ghrelin 10 nM and 100 nM, respectively, in the presence of the absence of extracellular calcium; Figure 5C). Moreover, JMV1843 (100 nM) was unable to induce any significant [Ca 2+ ]i response (P = 0.44 versus basal, P = 0.007, as compared to the effect of JMV1843, in the presence of extracellular calcium; Figure 5D). By contrast, under the same experimental conditions, the metabotropic glutamate 1/5 receptor agonist DHPG still triggered robust [Ca 2+ ]i transients (P < 0.001, as compared to basal; Figures 5C and5D). GHS-R1a activation thus mainly produced intracellular Ca 2+ increase in hippocampal neurons by promoting Ca 2+ influx from the extracellular medium. In our hands, L-type VGCC largely mediates the Ca 2+ influxes observed after depolarization in hippocampal cultures [START_REF] Pringos | Synthesis and characterization of a cyclooctapeptide analogue of omega-agatoxin IVB enhancing the activity of Cav2.1 calcium channels activity in cultured hippocampal neurons[END_REF]. Thus, verapamil, a L-type VGCC blocker was tested. In the presence of verapamil (50 µM), the ghrelininduced [Ca 2+ ]i increase was almost fully inhibited (P < 0.001, as compared to the effect of ghrelin, in the absence of verapamil; Figure 5E), although a residual K + -mediated [Ca 2+ ]i rise could still be evidenced (P < 0.001, as compared to the effect of K + in the absence of verapamil; Figure 5F).

As we found that GHS-R1a stimulation produced a strong potentiation of the glutamatergic AMPA receptor-mediated synaptic transmission in hippocampal slices likely via postsynaptic mechanisms, we next evaluated whether GHS-R1a could directly modulate the activity of ionotropic glutamate receptors, i.e. AMPA/KA and NMDA receptors. In order to test this hypothesis, cells were pre-incubated for 30 min with 100 nM JMV1843 before the application of either kainate (20 µM) or NMDA (10 µM). Both ionotropic glutamate receptor

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agonists induced a sustained increase in [Ca 2+ ]i (P < 0.001, as compared to basal).

Pretreatment with JMV1843 significantly enhanced kainate-mediated [Ca 2+ ]i changes (F 1,10448 = 21, P < 0.001; Figure 6A). Holm-Sidak post-hoc analysis indicated a significant difference (P < 0.0005) for data points obtained between time 2 and 3 min. Conversely, a similar pretreatment significantly decreased NMDA-elicited [Ca 2+ ]i rises (F 1,11248 = 134, P < 0.001; Figure 6B), with a significant effect (P < 0.0005) for data points obtained between time 2 and 4 min.. This confirmed that ghrelin could modulate synaptic transmission by potentiating AMPA/KA receptor-associated physiological responses independently of NMDA receptor activation.

DISCUSSION

Recent studies showed that the orexigenic peptide ghrelin induced a strong modulation of hippocampal function which may be involved in the positive effect on cognition and learning mediated by hunger [START_REF] Carlini | Ghrelin increases anxiety-like behavior and memory retention in rats[END_REF]Diano et al., 2006;[START_REF] Olszewski | Ghrelin in the CNS: from hunger to a rewarding and memorable meal?[END_REF][START_REF] Andrews | Central mechanisms involved in the orexigenic actions of ghrelin[END_REF]Davis et al., 2011). However, the mechanisms involved in this potentiation are not completely deciphered as ghrelin may activate a plethora of molecular mechanisms. Here, we show on rat hippocampal slices that ghrelin potently enhances synaptic transmission by activating PKA and ERK pathways. This effect of ghrelin results from the activation of GHS-R1a, as evidenced with selective ligands, i.e. the agonist JMV1843 and the antagonist JMV2959.

Ghrelin signaling in the hippocampus is often related to the induction of PKA pathway. Indeed, activation of PKA signaling occurred in organotypic hippocampal slices incubated with relatively higher concentrations of ghrelin (from 50 nM to 1 µM), as compared to those used herewith. This activation resulted in the phosphorylation of CREB, and in fine, in the

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This article is protected by copyright. All rights reserved. phosphorylation of the GluN1 subunit of the NMDA receptor [START_REF] Cuellar | Ghrelin-induced activation of cAMP signal transduction and its negative regulation by endocannabinoids in the hippocampus[END_REF]. As far as long-term mechanisms of potentiation are concerned, we find here that the stimulation of both PKA and ERK pathways are critical steps in the ghrelin-induced long-lasting synaptic enhancement in the CA1 area of the hippocampus. It is known for long that PKA activation is crucial to trigger consolidated synaptic plasticity produced either electrically or chemically [START_REF] Kandel | The molecular biology of memory storage: a dialogue between genes and synapses[END_REF]. ERK phosphorylation downstream PKA activation is also required for the consolidation of synaptic plasticity in many experimental models as previously reported [START_REF] Kanterewicz | The extracellular signal-regulated kinase cascade is required for NMDA receptorindependent LTP in area CA1 but not area CA3 of the hippocampus[END_REF][START_REF] Lynch | Long-term potentiation and memory[END_REF][START_REF] Sweatt | Mitogen-activated protein kinases in synaptic plasticity and memory[END_REF]. It is noticeable that activation of PI3Kinase-Akt signaling in the dentate gyrus may also stand for the occurrence of ghrelinevoked hippocampal synaptic plasticity [START_REF] Chen | Local infusion of ghrelin enhanced hippocampal synaptic plasticity and spatial memory through activation of phosphoinositide 3-kinase in the dentate gyrus of adult rats[END_REF], and also most likely for the enhancement of food-intake induced by intra-hippocampal infusion of ghrelin [START_REF] Kanoski | Ghrelin signaling in the ventral hippocampus stimulates learned and motivational aspects of feeding via PI3K-Akt signaling[END_REF]. Thus our data further add to the complexity on the intracellular pathways activated by ghrelin in central neurons.

Contrasting results regarding the involvement of NMDA receptor activation in ghrelinmediated effects have been reported so far. In organotypic culture of hippocampal slices, NMDA receptor blockade actually inhibited the ghrelin-induced CREB activation [START_REF] Cuellar | Ghrelin-induced activation of cAMP signal transduction and its negative regulation by endocannabinoids in the hippocampus[END_REF], as well as AMPA receptor insertion [START_REF] Ribeiro | Ghrelin triggers the synaptic incorporation of AMPA receptors in the hippocampus[END_REF], suggesting that ghrelin could induce the potentiation of excitatory synaptic transmission via an enhancement of NMDA receptor activity. By contrast, in vivo, the ghrelin-mediated enhancement of excitatory synaptic transmission in the dentate gyrus was insensitive to NMDA receptor blockade [START_REF] Chen | Local infusion of ghrelin enhanced hippocampal synaptic plasticity and spatial memory through activation of phosphoinositide 3-kinase in the dentate gyrus of adult rats[END_REF]. Using both acute hippocampal slice preparations and primary neuronal cultures, our data show an interaction between ghrelin-induced potentiation of excitatory synaptic transmission and NMDA receptor activation. Indeed, we firstly observed that NMDA receptor blockade enhanced the ghrelin-mediated synaptic potentiation in slices,

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This article is protected by copyright. All rights reserved. and secondly that ghrelin decreased the NMDA receptor-associated Ca 2+ influx in primary hippocampal neuron cultures.

The discrepancies in NMDA receptor dependency could be related to the experimental models used and/or to the metabolic environment of the neurons encountered in these preparations [START_REF] Yang | Hunger States Switch a Flip-Flop Memory Circuit via a Synaptic AMPK-Dependent Positive Feedback Loop[END_REF]Haam et al., 2014). Indeed, the effect of ghrelin on synaptic transmission in the hypothalamus is linked to the activity of the AMP activated kinase, a sensor for cellular metabolic status. Similarly, as reported in NPY-positive hypothalamic neurons, ghrelin-induced [Ca 2+ ]i changes require AMP activated kinase activity [START_REF] Kohno | Ghrelin raises [Ca2+]i via AMPK in hypothalamic arcuate nucleus NPY neurons[END_REF].

On the other hand, we found that GHS-R1a activation reinforced AMPA receptormediated responses in hippocampal neurons and that the paired-pulse facilitation ratio was unchanged after ghrelin treatment, consistent with the prominent recruitment of postsynaptic mechanisms for obtaining the enduring potentiation of synaptic transmission [START_REF] Ribeiro | Ghrelin triggers the synaptic incorporation of AMPA receptors in the hippocampus[END_REF]. Our data therefore support the possibility that ghrelin-induced potentiation relies upon an enhancement of AMPA receptor-mediated synaptic transmission by promoting the postsynaptic insertion of AMPA receptors.

In the CA1 area of acute hippocampal slices, the ghrelin-induced long-lasting enhancement of synaptic transmission that we observe here is reminiscent of forms of longterm potentiation independent of NMDA receptors and dependent on PKA activation, which have been identified in CA3 [START_REF] Weisskopf | Mediation of hippocampal mossy fiber long-term potentiation by cyclic AMP[END_REF] but also in CA1 [START_REF] Lante | Developmental switch from LTD to LTP in low frequency-induced plasticity[END_REF][START_REF] Lante | Unveiling novel forms of hippocampal synaptic plasticity with microelectrode arrays[END_REF] areas of the hippocampus, as well as in amygdala [START_REF] Huang | Low-frequency stimulation induces a pathway-specific late phase of LTP in the amygdala that is mediated by PKA and dependent on protein synthesis[END_REF].

The ghrelin-induced activation of PKA may initially result from an increase in [Ca 2+ ]i and subsequent stimulation of Ca 2+ -dependent adenylyl cyclases [START_REF] Chetkovich | N-methyl-D-aspartate receptor activation increases cAMP levels and voltage-gated Ca2+ channel activity in area CA1 of hippocampus[END_REF] to produce cAMP. Indeed, GHS-R1a may couple Gq [START_REF] Mousseaux | Regulation of ERK1/2 activity by ghrelin-activated growth hormone secretagogue receptor 1A involves a PLC/PKCvarepsilon pathway[END_REF][START_REF] Lau | The constitutive activity of the ghrelin receptor attenuates apoptosis via a protein kinase C-dependent pathway[END_REF]

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This article is protected by copyright. All rights reserved. [START_REF] Sun | Growth hormone secretagogues reduce transient outward K+ current via phospholipase C/protein kinase C signaling pathway in rat ventricular myocytes[END_REF] and subsequently activate PLC and mobilize Ca 2+ from inositol-1,4,5trisphosphate (IP 3 )-sensitive stores [START_REF] Camina | Regulation of ghrelin secretion and action[END_REF]Falls et al., 2006). We verified that ghrelin and JMV1843 actually triggered increases in [Ca 2+ ]i in hippocampal neurons. More precisely, we found here that [Ca 2+ ]i changes elicited by GHS-R1a activation mainly resulted from influxes, as no [Ca 2+ ]i changes could be observed in the absence of extracellular calcium. Moreover, we observe here that ghrelin-induced [Ca 2+ ]i rises almost exclusively depend on L-type Ca 2+ channels opening as evidenced by the strong inhibitory action of verapamil, a L-type Ca 2+ channel blocker. Interestingly, the requirement of extracellular Ca 2+ for ghrelin to elicit [Ca 2+ ]i increases and to produce its neurophysiological effects has also been evidenced in neuronal populations involved in food intake and neuroendocrine regulations. For instance, ghrelin induces [Ca 2+ ]i increases dependent on both extracellular Ca 2+ and PKA activation in Neuropeptide Y containing neurons of the arcuate nucleus of the hypothalamus [START_REF] Kohno | Ghrelin raises [Ca2+]i via AMPK in hypothalamic arcuate nucleus NPY neurons[END_REF]. Similarly, ghrelin triggers [Ca 2+ ]i rises and growth hormone release in an extracellular Ca 2+ dependent manner in goldfish pituitary cells (Grey & Chang, 2009). Thus, in these neuronal populations, ghrelin-associated signaling seems to mainly involve Ca 2+ influxes through voltage-gated channels, rather than Ca 2+ mobilization from internal stores. This is further supported by the fact that, in these experimental models, ghrelin-mediated [Ca 2+ ]i rises are sensitive to N-type [START_REF] Kohno | Ghrelin raises [Ca2+]i via AMPK in hypothalamic arcuate nucleus NPY neurons[END_REF] or L-type (Grey & Chang, 2009) Ca 2+ channel blockade, respectively. Moreover, Ca 2+ channel opening is required for observing excitatory effects of ghrelin on hypothalamic GHRH neurons [START_REF] Osterstock | Ghrelin stimulation of growth hormonereleasing hormone neurons is direct in the arcuate nucleus[END_REF]. By contrast, blocking IP3 receptors with xestospongin-C does not seem to alter ghrelin-mediated CREB activation in CA1 hippocampal neurons [START_REF] Cuellar | Ghrelin-induced activation of cAMP signal transduction and its negative regulation by endocannabinoids in the hippocampus[END_REF].

The intracellular Ca 2+ changes elicited by ghrelin could thus result from the activation of voltage-gated Ca 2+ channels subsequent to membrane depolarization. In this line, rapid

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depolarizing responses to ghrelin have been observed in various neuronal subtypes, including substantia nigra, dorsal root ganglion and CA1 hippocampal [START_REF] Shi | Peptide hormone ghrelin enhances neuronal excitability by inhibition of Kv7/KCNQ channels[END_REF], as well as cerebellar [START_REF] Sun | Ghrelin suppresses Purkinje neuron P-type Ca(2+) channels via growth hormone secretagogue type 1a receptor, the betagamma subunits of Go-protein, and protein kinase a pathway[END_REF] neurons. Ghrelin enhances spike frequency likely by inhibiting voltage-gated K + channels, as recently described in CA1 hippocampal neurons [START_REF] Shi | Peptide hormone ghrelin enhances neuronal excitability by inhibition of Kv7/KCNQ channels[END_REF]. Therefore, long-lasting potentiation of excitatory synaptic transmission may be initiated by the depolarizing action of ghrelin, which leads to the activation of intracellular pathways resulting in the onset of consolidated enhancements of excitatory transmission.

LEGENDS FOR THE FIGURES

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This article is protected by copyright. All rights reserved. ghrelin, as indicated by numbers. In the presence of JMV2959, ghrelin did not elicit any significant modification of synaptic transmission (P = 0.34, as compared to basal transmission). (B). Effect of the specific GHS-R1a agonist, JMV1843 (100 nM) on excitatory synaptic transmission (n = 9). Graphs are illustrated by representative traces extracted before and after the application of JMV1843 as indicated by numbers.

Figure 1 :

 1 Figure 1: Action of ghrelin on CA1 synaptic transmission. (A) Long-lasting enhancement of

Figure 2 :

 2 Figure 2: Pharmacological characterization of the ghrelin action on excitatory transmission in

Figure 3 :

 3 Figure 3: Effect of ghrelin on synaptic transmission under NMDA receptor blockade. The

Figure 4 :

 4 Figure 4: Intracellular pathways involved in the ghrelin-mediated enduring synaptic

Figure 5 :

 5 Figure 5: Intracellular [Ca 2+ ]i changes induced by either ghrelin or JMV1843 in cultured

Acknowledgements:

This work was supported by grants from CNRS and the French Ministry for Higher Education and Scientific Research. The authors have no conflict of interest of any kind to declare.

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This article is protected by copyright. All rights reserved. mediated [Ca 2+ ]i rise. Control experiment (E) was performed by applying ghrelin (100 nM) and then K + (30 mM) (n = 120 cells). Verapamil (F) was applied 2 min prior to ghrelin and K + (n = 95 cells). Data presented in E and F was obtained from cells grown on different coverslips. Each drug was applied for 1 min at the indicated concentrations. [Ca 2+ ]i changes mediated by either 20 µM kainate (A) or 10 µM NMDA (B) were measured in neurons pretreated (filled circles) or not (open circles) with the specific GHS-R1a agonist, JMV1843 (100 nM for 30 min). Glutamate receptor agonists were applied for 1 min. On the graphs, data obtained from pretreated cells have been superimposed with those obtained from their corresponding control cells without pretreatment. In (A) data are averages of ratios from 257 and 101 cells for control and pretreated cultures, respectively. In (B) data are averages of ratios from 241 and 209 cells for control and pretreated cultures, respectively. Data have been normalized against the fura2 ratio measured in control cells prior to any treatment. * indicates P < 0.001 and ** indicates P < 0.0005 when comparing data from control vs pretreated cells.

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Ghalib Shiraz Dhalla's Ode

Lata Sandeep Bakshi

Bollywood Homes: Queer Diasporic Identity in

Keywords: queer diasporas, South Asian diasporas, Bollywood, South Asian cultures and race, home, Ghalib Shiraz Dhalla

Examining queer cultural practices in the articulation of diasporic subjectivity, this article problematises a redemptive reading of queer diasporas. Through a close reading of Ghalib Shiraz Dhalla's first novel Ode to Lata, I underscore the significance of disputing logics of suppressing female sexual subjectivity in queer discourses and concurrently challenge the entrenched racial hierarchies in South Asian diasporic populations. Placing the (male) queer diasporic subject at the centre of debates on home, race and queerness extends the critical span of queer and diaspora studies. It enables multiple interpretations of conventional readings of both queerness and diasporas.

racial and sexual position of gay male diasporic subjectivity in Ghalib Shiraz Dhalla's first novel Ode to Lata (2002). Dhalla's narrative is significant in the ways in which it disputes conventional conceptions of diaspora as theorised from a heteronormative perspective by challenging existing dominant paradigms of home. South Asian queer diasporic subjectivity recasts the vexed site of home as the repository of same-sex desire. However, the articulation of the queer diasporic male identity is dependent upon the simultaneous construction of embedded racial binaries and the elision of female sexual subjectivity. Although through explicit (re-)appropriation of film songs the novel highlights queer diasporic practices/memory of reading Bollywood as a specifically queer cultural export, I suggest that Dhalla rehearses Bollywood's uncritical obsession with Euro-American racialised conceptions of beauty to construct the protagonist Ali's queer identity.

As a theoretical framework, I extend Gayatri Gopinath's formulation of the queer diasporic frame of analysis that signals towards 'alternative forms of collectivity and communal belonging that redefine 'home' as national, communal, or domestic space outside a logic of blood, purity, authenticity, and patrilineal descent ' (2005a: 158). Combining insights from studies of diaspora and queer theory to unravel the interconnection between diasporic formations and queer cultural practices, I place the queer diasporic subject at the centre of discourses on queerness, diaspora, race and home. Such an approach undermines the rigid hierarchy that locates non-white queer subjects in peripheral boundaries and simultaneously addresses the absence of race as a useful category of analysis in postcolonial studies. By centring queer cultural practices such as Bollywood in diasporic narratives of home, history and belonging, this critique offers a new perspective on both queerness and traditional heteronormative frames of diaspora. As Gopinath suggests, 'suturing 'queer' to 'diaspora' points to those desires, practices, and subjectivities that are rendered impossible and unimaginable within conventional diasporic and nationalist imaginaries ' (2005a: 158). In this essay, I engage with those alternative forms of belonging and desire that suture together queer cultural practices to diasporic identities.

Queer diasporic homes

Dhalla is an Indian-Kenyan queer migrant to the United States. Written in the first-person voice, Ode to Lata, places the queer diasporic subject at the centre of issues of migration, home and diaspora. The novel is significant in the ways in which it represents the homosexuality of its protagonist, Ali, and highlights competing claims to his Indian, Muslim, Kenyan Asian, diasporic identity in his new home Los Angeles. It parallels Ali's loss of and longing for home with his yearning for his lost lover, Richard. The alternating appearance of Richard and Kenya attests to the impossibility of their separation in Ali's consciousness and suggests a nostalgic framework of longing for Richard and belonging to Kenya. Although Ali attempts to construct his American identity and feel comfortable in it by regulating his life to the rhythms of American television, the constant eruptions of his Kenyan past and his Indian culture create a sense of loss, nostalgia and longing.

Dhalla's novel rewrites conventional accounts of home in diasporic imagination from the perspective of the queerness of its protagonist. In relation to the multiple definitions of home in diasporic discourses, Avtar Brah points out that ''home' is a mythic place of desire in the diasporic imagination ' (1996: 188). The construction of the imaginary geographical homeland of Kenya illustrates a mnemonic process, which becomes both parallel and similar to Ali's desire for Richard. Kenya appears as an imaginary location to which Ali returns as he constructs fictions of his past. At the beginning of the novel, for Ali, 'the image of the Kenyan flag with its brilliant red and green colors' fades in comparison to the 'impregnable promise of benevolence' exemplified by the American flag (2). However, as the novel progresses, Kenya becomes a fiction that like Richard, unfolds within the confines of his imaginary belonging.

The nostalgia for Kenya is charted in fictional reconstructions whereby he recreates the Kenyan railway and his attachment to it: 'I had freedom in geography only to be forever captured by the memories of the home I left behind. In my dreams, I still ride the railway' (4). Although these events once existed in the realm of reality, their nostalgic invocation locates them within the boundaries of his imagination.

For Ali, home represents an intractable link to non-normative desire. Homecoming in the novel re-signifies home which conventionally functions as a site of heteronormativity that obliterates queerness. The concept of home is a contested terrain in scholarship on diaspora. Home, family, homeland, community and nation assume the status of problematic sites, which reproduce dominant structures and reinforce their mutual legitimacy. Following scholarly work on the interconnectedness of home, family, community and the nation, feminist critiques of the nation examine the position of women in discourses of family and home. Inderpal Grewal and Gaurav Desai, for instance, assert the centrality of women as emblems of national identity through their role in home and family, whilst Sondra Hausner and Jane Garnett discuss the relationship of religion with diasporic belonging [START_REF] Grewal | Home and Harem: Nation, Gender, Empire and the Cultures of Travel[END_REF]: 7, Desai 2017: 333, Hausner and Garnett 2016: 1). Within lesbian and gay discourses home typically functions in opposition to queerness and migration (away from home) represents a vital component of queerness. In this scheme, Ali's migration to Los Angeles renders Kenya by contrast as a 'home' of compulsory heteronormativity. However, Ali's brief return to Kenya and his constant nostalgic longing problematise any neat assessment of 'home' as a suppressor of queerness. On his visit to Kenya, Ali meets his bisexual lover Nawaz and even though Nawaz is awaiting his marriage, Ali feels 'a desire so urgent, it was acutely painful' and the 'familiar rush pulsating through my body' (94).

In her work on queer migrations, Anne-Marie Fortier challenges the 'tendency to oppose queerness and the childhood home, where the latter is a space where queerness does not fit (2003: 116).' Similarly, Gopinath provides an elucidatory account of the function of home in queer diasporic texts. She explains, queer diasporic texts … lay claim to both the space of 'home' and the nation by making both the site of desire and pleasure in a nostalgic diasporic imaginary. The heteronormative home, in these texts, unwittingly generates homoeroticism. This resignification of 'home' within a queer diasporic imaginary makes three crucial interventions: first, it forcefully repudiates the elision of queer subjects from national and diasporic memory; second, it denies their function as threat to family/community/nation; and third, it refuses to position queer subjects as alien, inauthentic, and perennially outside the confines of these entities. (2005b: 15) For the South Asian queer subject, the heteronormative home, as Gopinath suggests, by generating homoeroticism becomes a signifier of same-sex desire. If Kenya represents a phantasmatic place, it also represents the possibility of queer belonging apparent in the dramatic reliving of Ali's sexual past as he awaits sexual deliverance from Nawaz. His Kenyan home becomes the signifier of his adolescent queerness. It exemplifies David Eng's formulation of 'horizons of being and becoming ' (2010: 183). The resignification of home as a location of queer pleasure in Ali's imaginary disputes the claim that home can only be a place where queerness and non-normative desires exist as outsiders.

Crucially, Ali's encounter with Nawaz in Kenya serves to interrogate the assumption of nonwestern (queer) sexual experiences as either less emancipated or marginal. Ali shifts from a position of the newly acquired imperialist gaze to an acknowledgement of the complexity of sexuality in Kenya. Nawaz's impending marriage initially prompts Ali to look at Nawaz's covert bisexuality from his position of privilege as an American émigré. Contrasted to the discourses of American queer identity politics, Kenyan sexual experiences appear simplistically uncategorised, evident in Nawaz's engagement or later his friend Akil's 'imposed' marriage (107). However, Ali soon realises that American and Kenyan attitudes to homosexuality are not constitutive of a binary hierarchy. The novel contests the go-west narrative of queer diasporic subjects to find sexual freedom by re-energising Ali's desire for Nawaz in spite of his contempt for the latter. After the sexual encounter with Nawaz, Ali wonders who had 'made the right choice' (100). His own sexual discontentment in Los Angeles reminds him of the inefficacy of his choice. He concedes that 'perhaps labels are truly for cans of food. Not people. That we were all simply sexual beings' (99). Ali's statement provides an effective counterpoint to the homonormativisation of 'gay' identities based on the US frame of identity classification. It points to the universalising tendency of global models at the expense of local experience.

Critiquing conventional diasporic narratives, Gopinath observes that diaspora traditionally refers 'to a system of kinship reckoned through men' (2005b: 5). However, the mother-son diasporic bond in Ode to Lata is an exception in literatures of diaspora. Ali's father, like Richard, is an absent figure. Ali identifies with his mother, whereas Richard and his lovers stand for the lost father figure. He relates to 'the psyche of his dramatic Indian mother' and, wants to 'fuse back into her. To become one with her. To be her ' (45, 130). Like his mother, he recasts himself as 'forgiving, enduring and perhaps even preventing him from straying again' (39). By presenting Ali's identification with his mother, Dhalla repositions the mother-son attachment as a primary site of affiliation within the diaspora. Further, his purportedly feminine subject position, implied by this identification, disrupts the logic of gender fixity whereby adult males must identify with masculine role models.

Creating an intergenerational connection, Bollywood opens up alternative ways to account for diasporic identity, which does not rely solely on geographical links. Studies of the Jewish diaspora suggest that generational links between Jewish peoples de-emphasise the significance of geographical territory as the basis for thinking about Jewish identity. Jewish culture becomes a critical site that disassociates the natural bond between 'this people and a particular land' (Boyarin and Boyarin 2002: 108, see also , Braziel 2008: 13-17). Similarly, Bollywood represents a cultural form that privileges the generational over the geographical link in the novel. The prominence of the product of export that Bollywood films signify illustrates the cultural attachment of Ali and his mother to India, a country in which they have never lived.

The title of the novel invokes the renowned Bollywood 'playback singer' Lata Mangeshkar, 1 and draws attention to the significance of Bollywood in the narrative. Ali finds himself elated when listening to Bollywood music while his mother relates it to the past, 'those days' with his father (149, emphasis in original). As a child in Kenya, Ali accompanied his mother to the screening of Bollywood films, which was like a ritual for the 'entire Indian community of Mombasa' who 'would prepare for the excursion as early as noon by packing tiffins of Indian viands' (150). Bollywood thus creates a generational link rather than a geographical link between diasporic subjects. Ali's migration from Kenya to America is an example of geographical dislocation. However, in terms of his attachment to Bollywood, his Indian identity exemplifies the particularities of cultural displacement.

Queer Bollywood homes

In implicit relation to home as an imaginary construct in Dhalla's narrative, Bollywood recalls the nostalgic homing desire of South Asian diasporic populations. Jigna Desai notes 'the increasing centrality of diaspora and the transnational class to the postcolonial nation-state due to the deterritorialization of the nation and other global processes ' (2004: 184). Bollywood produces a fictional version of home, nation and nationalism through cinematic representation by representing the 'nation as a mythical community' [START_REF] Virdi | The Cinematic ImagiNation: Indian Popular Films as Social History[END_REF]: 1, see also, Prasad 2000: 28). It represents the anxiety surrounding the cosmopolitan, globalised image of India that the postcolonial nation aims to proliferate (Rai 2009: 30-32). Within diasporic circles, Bollywood constitutes a cultural tie with the lost homeland. As Ziauddin Sardar remarks, Indian films, the prime 'cultural referent' in Asian Britain, provide 'a direct emotional link with the subcontinent ' (1998: 22). Virdi similarly contends that Bollywood 'resonates powerfully with the Indian diaspora, often becoming their only connection with the homeland and the main intergenerational culture diasporic families share' (2003: 2). For Ali, Bollywood represents a cultural conduit for his connection to an 'Indian' past in Kenya. It connects Ali to the other members of the Indian diaspora-Farida, Salman and Riyaz in Los Angeles.

Bollywood enables Ali to express his feelings about Richard in a Western context where men's expression of emotion is taboo since feelings 'feminise' men. Scholars such as Lalita [START_REF] Gopalan | Cinematic Interruptions: Action Genres in Contemporary Indian Cinema[END_REF] and Vijay [START_REF] Mishra | Bollywood Cinema: Temples of Desire[END_REF] show that as a paradigm of the interplay of sexual and gender tropes, Bollywood opens a cultural space for cross-gender and non-normative sexual identification. Emotional excess, traditionally associated with women in the West, serves to heighten the loss of Richard and gives a tragic expression to Ali's feelings. The first word of the title Ode, as explained in Webster's dictionary, refers to a lyric poem, which is marked by exaltation of feeling and style. Bollywood's exalted emotions and melodrama are reinforced if read in conjunction with the definition of the literary style of an ode. Additionally, the queer diasporic subject and his attachment to Bollywood decentre the global hegemony of American culture. By aligning Ali and Salman to the melodies of Lata Mangeshkar, Dhalla rewrites the identificatory practices of 'a new generation of Indians that had never been to India; Indians who had become multicultural ' (190). The resignification of Bollywood as a cultural practice, at the centre of identificatory structure, serves two purposes. It creates a world where 'Lata Mangeshkar ousted Barbara Streisand' such that local cultural practices precede dominant/global ones (176). The global becomes a site of contestation where different local sites interact in an equable relation. Second, it brings to the fore and emphasises the queer subtext of Bollywood, claiming it as a diasporic queer cultural practice.

Ali's identification with Bollywood actresses Rekha, Meena Kumari and the performer Helen reveals a potential for cross-gendered queer identification. Bollywood functions as a critical site that resonates with queer modes of gender and sexual non-normativity. Even though Bollywood articulates an anxiety about securing national, cultural and sexual borders through narratives of cultural purity and authenticity, critical analyses have sought to read the faultlines, which disrupt discourses of a coherent heterosexuality or national community. The focus on song-and-dance performances in Bollywood films, oft regarded as unnecessary to the plot, uncovers a thematic trope of erotic visual pleasures. Shohini Ghosh remarks that 'as powerful vehicles of emotions and aspirations, songs and dances often play out Mikhail Bakhtin's notion of the carnivalesque ' (2002: 211). As an embodiment of the 'carnivalesque,' song-and-dance sequences disrupt traditional hierarchies of class, gender or sexuality. Vanita suggests that 'while the plot of the films is heavily didactic and censorious of erotic pleasure, the half-dozen songs that punctuate every film tend to celebrate erotic life, including illicit eroticism' (2005:

288).

Ali's construction of his queer subjectivity through identification with Bollywood songs highlights the queer appropriation of a dominant cultural expression. The songs are particularly significant for queer subcultures because marginal or unconventional unions find expression in them. These bonds include cross-class love, inter-religious marriage and, to a certain extent, male and female homoeroticism. As Vanita explains, 'many songs, especially older ones, are in the first and second person ('I love you') and thus carefully avoid gendering either singer or addressee.' She adds that the strategy to leave the gender open to interpretation also functions through the use of 'the optative voice and the plural' (2005: 283). Ali's performance of Helen's song 'Piya tu Ab to Aaja' ('O my lover, please come Now') from the film Caravan (dir. Nasir Hussain, 1971) is exemplary of the gendered irresoluteness of the lyrics. Although sung as a heterosexual cabaret duet, it can successfully describe any gendered position since the song, carefully omits any reference to the gender of the singer. By avoiding any reference to the singer or the addressee, the song can be sung by both the genders. Sexual desire becomes visible via an un-gendered position and can therefore incorporate non-normative identification.

Ali's narcissistic cross-gendered identification with the female singer challenges the norms of gender identification like the expression of emotion afforded by the songs. Additionally, his reappropriation of the song queers the most important cultural export of India, Bollywood. Ali's performance of Helen's song opens the possibility of queer self-fashioning in South Asian cultures in which Bollywood appears as a mode of expression where queer desire can be made intelligible.

Ali's identification with Helen further underscores the solidarity of marginal identities.

Belonging to a non-Hindu/Anglo-Indian minority, Helen offers the most direct contrast to the idealised, traditional heroine of Indian films who occupies the role of the submissive, asexual subject position. Helen's hyper-sexualised performances function as a 'clutter of signifiers of Westernization,' often involving sexual licentiousness (Thomas 1989: 11). They define, by contrast, the cultural 'purity' of the Hindu/Indian heroine. As the racialised and religious outsider, Helen embodies the threat of corruption of the national body, which is symbolised by the non-Westernised and sari-clad Hindu/Indian woman. By making Helen a pivotal point in Ali's childhood narrative, Dhalla locates his queer diasporic subject in an alliance with other marginalised identities that challenge the fiction of a unified Indian culture. Ali's other strong identification with Bollywood icon Meena Kumari also places the marginal subject at the centre of discursive representation. Meena Kumari's portrayal of a courtesan in the film Pakeezah (The Pure One, 1971) has acquired an iconic status in Bollywood spectatorship. Ali suggests that one particular song from the film, in which Meena Kumari declares that she has been sexually exploited by all the members of the society from the postman to the policeman, is 'one of the campiest songs' (62). For him, 'this should have been the litmus test for any Indian parent to discover a son's homosexuality. The infamous Pakeezah song' (62). Like Helen, Meena Kumari represents the 'other' as the degenerate 'Muslim' tawaif (courtesan). However, the film condemns the society for its treatment of the prostitute, who remains 'the pure one'.

Ali's identification with Meena Kumari thus conjoins queerness with stigmatised identities and calls into question the cultural stigmatisation of marginal subjects and non-normative desire.

Queer male subjects, elision of female sexuality and whiteness

Paradoxically, the enabling queer diasporic identification with the oppressed female figure serves to reinforce male control over the female body in the novel. The glamorous figures of Helen and Meena Kumari efface the effects of exploitation of female bodies for male pleasure. Asha Kasbekar contends that:

the Hindi film upholds the patriarchally determined feminine idealization through inflated rhetoric on chastity within the narrative, but resists the very same feminine ideals by offering the woman as 'spectacle' in the song-and-dance numbers, both idealization and fetishisation being themselves products of patriarchy. (2001: 294) Additionally, Meg Wesling reassesses the enabling narrative of queer global mobility within diasporas as particularly disadvantageous to women who are forced into prostitution on transnational scale (2008: 37). Similarly, Ali's mother appears both as an idealised and fetishised woman in Ali's imagination. As the ideal Indian mother, Ali refuses to acknowledge his mother's sexuality while his father is positioned as an active sexual agent. His mother explains that her husband did not even have 'proper intercourse' with her when she became pregnant because he does not wish to 'defile her' (5). For Ali, she is the template of the Mother India figure who sacrifices her life for the social good. At the end of the novel, he realises that as a mother she had sacrificed all her life so that he can have a better life. He feels an 'appreciation for all she had endured' (213). The guilt generated by not being able to give her grandchildren results from his view of her as the ideal Indian mother. Simultaneously, his mother embodies the threat of female sexuality who like Helen transgresses the legitimate boundaries of social order by desiring her son in an oedipal-castrative-incestuous frame. In his dreams, he imagines her 'hands ... on her breasts as if priming a weapon of torture' (205, italics in original). The desired female bodies of his mother, Helen and Meena Kumari therefore operate in a patriarchal structure that they threaten to rend as active agents of sexual desire.

The privileging of Ali's identification with the courtesan and the performer in the narrative over the development of the female character Farida's lesbianism inevitably marginalises the queer female diasporic experience in the novel. Apart from his mother, Ali's Indian lesbian friend Farida is the only other female character in the novel. Gopinath argues that female homoeroticism is a problematic site in diasporic renditions of Bollywood films, pointing to the 'elision of queer female diasporic sexuality and subjectivity' within queer diasporic texts (2005b: 19). Farida's character and lesbian negotiation of Indianness remain underdeveloped in the novel. As mentioned above, female homosocial play is one of the key subtexts in certain Bollywood songs along with an enduring history within South Asian folk cultures. However, Dhalla elides such references to female homoerotic pleasure and, as a result, Farida appears as the 'social matriarch' who conforms to the stereotype of woman as mother (197). Although Farida organises meetings for the queer South Asian support group Saath (togetherness), her lesbian identity remains relatively unexplored in the novel. For Ali, instead of being an actively desiring subject, Farida, like his mother, represents the intrusive woman who harasses Ali 'with ten phone calls a day' (201). He disregards Farida's attachment to her lover Chastity as uninteresting 'pussy problems ' (193). Unlike the other male members of Saath who are 'far too simpleminded,' he thinks of Farida as a cunning female who is responsible for his boycott from the group' (203). Her character reifies the chasm between male and female homosexuality. She appears as the stereotypical power-hungry lesbian who threatens the order of their support group.

Another limitation of the male queer diasporic subject position and the gay recuperation of Bollywood icons is the resurrection of 'whiteness' as culturally superior and desirable. Helen, Meena Kumari and Rekha are all fair-skinned Bollywood stars in an industry, which relegates 'dusky' heroines like Smita Patil to the so-called art-house cinema. The South Asian queer project of exploding normative gender identifications becomes complicit in uncritically accepting fairness and whiteness as desirable attributes. In contemporary Bollywood, the fairness of the heroine in the songs is often accentuated by placing her at the centre of a dance troupe of Euro-American females. Also, the demand for fairness in men is increasing through the promotion of skin-lightening creams for men by using Bollywood male superstars like Shah Rukh Khan for publicity. As Ali admits in the novel, 'as South Asians away from Asia, and, more painfully, away from the countries of our childhood, we tried to recreate the norms of our culture ' (195). In developing Ali's racial prejudice, Dhalla upholds these very 'norms of our culture' to criticism.

In Ode to Lata, Bollywood's uncritical appropriation and obsession of imperialist racist binaries of skin colour doubles in the form of Ali's prejudice towards other racialised positions

The queer diasporic subject's interaction with the racial binary uncovers the fractures within a marginalised position. Ali's reaction to Bill's pejorative remarks reflects and reinforces his own racial bigotry, similar to, as I mentioned above, the coalitional bonds with the feminine that appear embedded in patriarchal logic. The novel critiques Ali's biased position by making his attempts to counter Bill as failed propositions. Moreover, when Bill suggests the repatriation of all black populations to 'go back to where you came from,' Ali remains composed even though this comment infuriates him (242). As he says, 'And I felt ashamed because the same remark coming from Bill, from someone I was sexually attracted to, didn't compromise my desire for him' (242). Over the course of time, Ali even becomes convinced of the validity of Bill's reactionary position: 'Bill's prejudice, I justified, even in all its repugnance, revealed an honesty that deserved both admiration and pity' (243). The mixed feelings of 'admiration and pity' make Bill's comments excusable and acceptable to Ali. Ali's endorsement of Bill's position reflects and reconfirms the conventional paradigm of black-as-inferior in South Asian cultures. His ultimate rejection of blackness is secured when, in an attempt to forget his loneliness, he urinates on a black man in a bar. As he reflects upon all his 'emotional betrayals,' he urinates and 'splashes over this boy's dark skin as he beats himself frantically' (280).

Although figured as a voluntary act demanded by the black boy, Ali associates it as an act of revulsion: 'Dear God, What am I doing? What have I done?' (280). Even though, for Ali, this is an act of misdirected vengeance on past lovers, it is surely an expression of his contempt for blackness as well.

The ending of the novel portrays Ali as the tragic figure of Bollywood films who remains solitary even though he craves for love. Two key events at the end of Ode to Lata, however, signal a move away from the over-arching thematic concern of longing in the narrative. First, shortly after Ali's resignation, 'a new, vibrant young woman who had just emigrated from India took over the post of South Asian coordinator' of the support group Saath (284). Given Ali's barely disguised misogynistic position throughout the story, the appointment of the 'vibrant young woman' to his post signifies the novel's way of challenging such prejudice.

Simultaneously, on an invitation from the group, Ali finally decides to get himself tested for HIV. This positive step mirrors his ultimate redemption from longing for Richard and is reinforced by his life-affirming realisation: 'Alone, perhaps, but not bitter. Never bitter. I want to continue to feel desire because I want to continue to live' (284). These incidents point to the several ways in which Ali comes to terms with absence and desire of not only Richard but also of home and his 'gradually disappearing' family (283).

In this essay, I have outlined the significance of locating the South Asian queer diasporic subject at the centre of debates about ethnicity, home and nostalgia, race, and diaspora. My interpretation of Dhalla's novel has departed from conventional readings of both queerness and diaspora to involve the complex matrices of race and ethnicity, which appear essential -even though they are invariably diminished -to the construction of queer diasporic identity. It has explained that the subversive possibilities of certain regimes of representation can repeat the norms of other forms of exclusion and a coalitional politics of subordinated identity categories is a crucial exercise, but a critical assessment of such alliances is equally relevant.

Dhalla's novel problematises male queer diasporic subjectivity whereby the simultaneous repulsion of, and attachment to the mother and to the country of origin function in parallel to other exclusionary practices. It complicates those singular readings of diasporic identities, which privilege the linear narrative of geographical displacement over and above other affiliative formations. Moreover, cultural products, like Bollywood, that become interstitial to queerness and diaspora replicate the dominant norms of racial hierarchies. I have highlighted the need to reassess such cultural practices by paying attention to their exclusionary effects. As I have argued, if Ode to Lata offers the recuperation of Bollywood as a queer cultural export for the diaspora, then it posits this diasporic queerness against stereotypes of black identities found in Bollywood. Concurrently, the articulation of queer male diasporic subjectivity invariably materialises through a literal erasure of female sexual desire. In this respect, male queer subject positions collude with heteronormative/nationalist ideologies. Boyarin, Daniel, and Jonathan Boyarin (2002) 'Diaspora: Generation and the Ground of Jewish Identity', in Anita Mannur & Jana Evans Braziel (eds.) Theorizing Diaspora: A Reader, Malden, MA: Blackwell Publishing, pp. 85-118. Brah, Avtar (1996) Cartographies of Diaspora: Contesting Identities, London: Routledge.

In Bollywood, a playback singer provides the voice to the on-screen actor. Lata Mangeshkar was awarded the prestigious award Bharat Ratna (the Gem of India) for her contribution to music and is the playback singer par excellence.

in Los Angeles. Discussions of inter-racial imbrication (between various ethnic/racial groups for instance) have often been neglected in postcolonial analyses. Malini Schueller, for instance, interrogates the elision of racial parameters in postcolonial theory, suggesting that diasporic subjects interact with the question of race in a complex grid wherein 'diasporas and transnational connections are significantly marked by the integument of race ' (2003: 36). In similar regard, the editors of the special issue of Social Text (1997) entitled 'Queer Transexions of Race, Nation and Gender' argue for the inclusion of race in queer theoretical frames because:

this deployment not only illuminates how various dimensions of social experience-race, sexuality, ethnicity, diaspora, gender-can cut across or transect one another, resulting in their potential mutual transformation; it also 'queers' the status of sexual orientation itself as the authentic and centrally governing category of queer practice, thus freeing up queer theory as a way of conceiving not just the sexual, but the social in general. (Harper et al. 1997: 1, emphasis in original).

Given the complexity of Ali's racialised position in Los Angeles, I suggest that the novel deploys queerness as a formulation of Ali's sexual as well as racial categorisation.

Mapping forms of imperial racist formulations, Ode to Lata offers an insightful account of the problematic issue of race within postcolonial theorisation. Ali's interaction with his only black gay friend, Dar, reveals a process of 'the ascendancy of whiteness,' to borrow a term from Jasbir Puar (2007: 2). For Ali, the maintenance of a safe distance from Dar not only repeats the imperialist trope of blackness-as-abjection, but crucially becomes an important factor in his association with whiteness. Puar argues that queer diasporic subjects are under duress to conform to standards of 'white' homonormativity such that they disassociate 'from others disenfranchised in similar ways in favor of consolidation with axes of privilege' (2007: 24).

Ali's reproduction of racism relies on such strategies of identification with whiteness. I contend that diasporic formations can subvert the dominant and simultaneously reinstate the notion of a return to the primal past. After their little outing to the gay bars of Los Angeles, Dar and Ali return to Ali's house. Ali gives him a separate glass of water and cannot overcome his racial barrier to sleep with Dar on the same bed. His racial prejudice in Los Angeles manifests itself as a return to his Kenyan past where 'black' is associated with inferiority. Friendship between Dar and Ali remains governed by the racial divide that separates them. Bollywood as a queer cultural practice incorporated by Ali in the construction of his queer subjectivity also informs the understanding of his own racial position as the non-white 'other' in America. The novel critiques Bollywood's racial prejudice and Ali's replication of it by making Richard unavailable to Ali. Ali yearns for Richard, whose Latino ethnicity (his surname is Lopez) still appears as racially 'white' in Ali's Kenyan Indian imaginary (21). In his first sexual encounter with a soldier in the novel, Ali is evasive about declaring his origins. He finds his Indian features 'embarrassing' and wants to pass as a Latino (21). As Ali says, 'Was this not a curse of every South Asian whose standards of beauty were in conflict with his own appearance' ( 22). Although as an explanation he points to the racial colonial constructs of white/non-white binary, the postcolonial Kenyan and Indian cultures continue and reinforce the inferiority of black skin: 'Imagine growing up in a country where being white automatically meant that you were entitled to the privileges that everyone had to struggle for' (23). Moreover, in his dream about his mother as a tormentor, Ali cannot imagine her as dark-skinned. He asserts that the woman in his dreams does not resemble his mother because, for him, his mother 'is certainly not as dark in complexion' in real life (204, emphasis in original).

Queer diasporas and race

Ali's brief encounter with Nelson, his black lover, further problematises cross-racial desire in the novel. By framing the Nelson/Ali relationship in a configuration of lust without emotion, Dhalla clearly disputes the racial bias of his protagonist. In his interaction with the racialised 'other', the South Asian queer diasporic subject reveals the limitations of cultural practices like Bollywood, which reinforce entrenched racial hierarchies. Nelson figures in the narrative as the signifier of rapacious sexuality. Jessie Daniels argues that the fear of black sexuality 'intensifies when it is combined with homophobia' (2016: 39). Nelson's 'bulbous cock bobbing between his legs' connects him to the stereotypical image of the black man reduced to his sexual prowess (116). Ali and Nelson's aggressive sexual encounters are contrasted with Ali's romantic longing for Richard. Equating black men with brutal sexuality, he prefers Nelson when the latter is demeaning and distant, when he is 'facing the back of my head in the grasp of his hand and grunting like a beast' (121, emphasis added). Further, he does not trust Nelson with his car and sinks his car keys in the toilet tank when he meets Nelson (115).

Michael Uebel claims that in the presence of the racial other 'the subject is intensely ambivalent, poised between desire and fear, incitement and interdiction, mastery and anxiety' (1997: 5). Certainly, Ali's suspicion of Nelson makes the latter more desirable: 'He aroused in me the kind of sexual compulsion that hits smack in the gut' (113). Even though Ali loathes his own ethnicity, he does not feel 'the need to conceal' his body from Nelson (117). Further, Ali uses Nelson as the final instrument in provoking the feeling of jealousy in Richard.

Ali's racial prejudice surfaces in his response to Nelson's infidelity. When Nelson and Ali's best friend, Adrian, have an affair, Ali refuses to forgive Nelson in contrast to his earlier clement behaviour towards Richard's betrayal. Compared to Adrian's 'little pale face, with the adoring amber eyes and prominent lashes, lashes that could make even his most venomous intentions seem benign,' Nelson is seen as a manipulator whose repentance is 'a cunning transference of blame' (164). Unlike Nelson, Ali pardons Adrian but given his complex relationship to whiteness, the forgiveness appears problematic. It functions as a medium to efface his sense of racial inferiority. By realising that Adrian is 'like the rest of us mortals,' both Ali and Adrian become equal in Ali's perception (171). Consequently, for Ali then, he belongs to the same world of whiteness that Adrian inhabits.

The contrast between Nelson and Bill, the hustler, reveals the racial fracture in the multicultural fabric of America. For Ali, Bill represents 'all the physical attributes of physical beauty' (220).

Like Richard, Bill is a Latino with an 'unjaded demeanor' (220). Although he is a hustler, Ali does not find any sign of 'contrived sexuality' in his eyes (220). Unlike with Nelson, there is an absence of animal imagery when Ali describes Bill. Their sexual encounter lacks the immediacy of the sexual act between Ali and Nelson. Instead of pursuing him, Ali wishes to 'elicit his desire for me' (228). Significantly, Bill exposes the racial fissures of American multicultural discourse. He has a tattoo on his arm, 'a derivative of Swastika,' which signifies his hatred of black people (241). As part Mexican and part American-Indian, Bill shares a similar history of oppression and displacement to that of the African Americans. However, he considers them in a frame of competition for equal rights where black people figure as obstacles. For him, the 'black monkeys' with their 'sorry-ass problems' always 'have more than we'll ever get from this country' (242). His racist comments place white America at the centre of rival ethnicities. Thus, the novel demonstrates that even though the multicultural bars of Los Angeles symbolise the entire multi-ethnic nation, the centrality of whiteness remains undisturbed by its absence in ethnic rivalry.


Upasna Sharma

Colin C Conine

Jeremy M Shea

Ana Boskovic

Alan G Derr

Xin Y Bing

Clemence Belleannee

Alper Kucukural

Ryan W Serra

Fengyun Sun

Lina Song

Benjamin R Carone

Emiliano P Ricci

Xin Z Li

Lucas Fauquier

Melissa J Moore

Robert Sullivan

Craig C Mello

Manuel Garber

Oliver J Rando
email: oliver.rando@umassmed.edu

Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals

Several recent studies link parental environments to phenotypes in subsequent generations. Here, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA levels in mature sperm, with decreased let-7 levels and increased levels of 5' fragments of glycine tRNAs. tRNA fragments are scarce in testicular sperm, but are gained as sperm mature in the epididymis. Epididymosomes -vesicles that fuse with sperm during epididymal transit -carry RNA payloads matching those of mature sperm, and deliver RNAs to immature sperm in vitro. Functionally, tRNA-Gly-GCC fragments repress genes associated with the endogenous retroelement MERVL, both in ES cells and embryos. Our results shed light on small RNA biogenesis, and its dietary regulation, during post-testicular sperm

SUPPLEMENTARY MATERIALS Materials and Methods

Figs.S1 to S13 Datasets S1 to S8 References (21) to (35) maturation, and link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.

RESULTS

There is accumulating evidence that parental environments can affect the health of offspring. For example, paternal nutrition influences offspring metabolism in mammals [START_REF] Rando | I'm Eating for Two: Parental Dietary Effects on Offspring Metabolism[END_REF]. Our prior published work showed that male mice consuming Low Protein diet fathered offspring exhibiting altered hepatic cholesterol biosynthesis, relative to offspring of Control males [START_REF] Carone | Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals[END_REF]. The mechanisms by which paternal conditions reprogram offspring phenotype remain elusive, as males can influence offspring via the sperm epigenome, microbiome transfer, seminal fluid signaling, or maternal judgement of mate quality ( 3 , 4 ). We therefore first tested whether metabolic gene expression was altered in offspring generated via in vitro fertilization (IVF) using sperm obtained from animals consuming Control or Low Protein (19% or 10% protein, respectively) diet. Despite the potential for IVF and embryo culture to obscure paternal effects on offspring metabolism, we nonetheless found that IVF-derived offspring of Low Protein males exhibited significant hepatic upregulation of the gene encoding the cholesterol biosynthesis enzyme squalene epoxidase, compared to Control IVF offspring ( 2 )(Fig. S1), demonstrating that paternal diet can affect offspring metabolism via information located in sperm.

As small RNAs are central to a broad range of epigenetic phenomena( 5 ), we isolated cauda sperm from males consuming Control or Low Protein diet, and purified small (<40nt) RNAs for analysis by deep sequencing. Resulting sequencing libraries reveal remarkably abundant (~80% of small RNAs) ~28-34nt tRNA fragments (tRFs), predominantly derived from the 5' ends of tRNAs (Fig. 1A-D,Tables S1-S2) [START_REF] Peng | A novel class of tRNA-derived small RNAs extremely enriched in mature mouse sperm[END_REF]. 5' tRFs are also abundant in cauda sperm from the bull B. taurus (Table S3), suggesting that tRNA cleavage in gametes is conserved among mammals, and perhaps more broadly [START_REF] Couvillion | A growth-essential Tetrahymena Piwi protein carries tRNA fragment cargo[END_REF]. Given the low RNA content of sperm relative to oocytes, we focus our analyses on highly abundant small RNAs in sperm. Low Protein diet affected levels of multiple small RNAs, including highly abundant tRNA fragments, across eight pairs of sperm samples (Fig. 1E-F). Most notably, 5' fragments of tRNA-Gly-CCC, TCC, and GCC exhibited a ~2-3-fold increase in Low Protein sperm, and tRF-Lys-CTT and tRF-His-GTG were similarly upregulated. In addition to tRFs, other RNA species differ in abundance between sperm samples, with several let-7 species being downregulated in Low Protein sperm.

We next assayed levels of intact tRNAs in testis, finding no correlation between dietary effects on testicular tRNA levels and tRF changes in cauda sperm (Fig. S2). This argues against the hypothesis that tRFs in mature sperm result simply from random degradation of tRNAs utilized during spermatogenesis. Moreover, deep sequencing and Northern blot analyses (Figs. 2A,C,S3,Tables S1-S2) revealed very low levels of tRNA fragments in testes or in various purified testicular spermatocyte/spermatid populations, raising the question of when sperm gain tRFs during maturation. After exiting the testis, sperm continue to mature for several days in the epididymis, and we find robust tRNA cleavage throughout this tissue (Figs. 2B,D,S4). Not only do overall tRF levels increase distally in the male reproductive system, but the spectrum of specific tRFs differs between testis, proximal caput epididymis, and distal cauda epididymis (Fig. 2D, Table S2).

Since our data suggest that small RNAs in mature sperm could have originated at multiple locations throughout the reproductive tract, we assessed the effect of paternal diet on small RNAs in testis (n=9 pairs), caput epididymis (n=6), and cauda epididymis (n=5) (Fig. S5). Intriguingly, two prominent dietary effects on the cauda sperm RNA repertoire -increased abundance of glycine tRFs, decreased abundance of let-7 -were recapitulated in the testis and epididymis, but not in liver, muscle, or blood (Table S1). Thus, tissues throughout the male reproductive tract -including mature sperm -exhibit consistent changes in glycine tRFs and let-7 in response to Low Protein diet, suggesting that similar diet-responsive pathways are present throughout the tract, and providing technical replication of the fundamental epigenomic changes wrought by Low Protein diet.

The finding of robust tRNA cleavage in the epididymis, but not testis, raises the possibility that the abundant tRFs in cauda sperm might be trafficked to sperm from the epididymal epithelium, rather than arising during testicular spermatogenesis. During transit through the epididymis, sperm fuse with small extracellular vesicles known as epididymosomes ( 8 -11 ).

To test the hypothesis that epididymosomes deliver small RNAs ( 12 , 13 ) to sperm, we purified epididymosomes (Fig. S6) and characterized their small RNA payload by deep sequencing. Epididymosomes carry high levels (~87% of reads) of 5' tRFs such as tRF-Glu-CTC and tRF-Gly-GCC, and small RNAs found in purified epididymosomes closely mirror (r = 0.96) those in cauda sperm (Figs. 2E,S6). Epididymosomal RNAs were resistant to RNAse treatment, and were found in epididymosomes from spermless Tdrd1 -/-mice, ensuring that vesicles purified from the epididymis are not generated from maturing sperm (Fig. S6G).

To further test the hypothesis that epididymosomes are responsible for shaping the RNA payload of sperm, we characterized small RNAs in sperm isolated from the proximal caput epididymis, finding that the RNA payload of caput sperm differs substantially from that of distal cauda sperm (Figs.3,S7) [START_REF] Nixon | The MicroRNA Signature of Mouse Spermatozoa Is Substantially Modified During Epididymal Maturation[END_REF]. Proximal-distal biases for specific tRFs along the epididymis were reflected in maturing sperm, showing a dramatic ~10-fold enrichment of tRF-Val-CAC, for example, in cauda relative to caput samples. To directly test whether epididymosomes can deliver their RNAs to caput sperm, we purified caput sperm and incubated them with cauda epididymosomes, then pelleted and washed resulting "reconstituted" sperm. Epididymosomal fusion with caput sperm was sufficient to deliver tRF-Val-CAC and other cauda-enriched tRFs to caput sperm in both mouse and bull (Fig. 3C-D) [START_REF] Caballero | CD9-positive microvesicles mediate the transfer of molecules to Bovine Spermatozoa during epididymal maturation[END_REF]. Taken together, these results are most consistent with a mechanism of RNA biogenesis in mammalian sperm in which tRFs generated in the epididymis are trafficked to sperm in epididymosomes, although they do not rule out the alternative hypothesis that intact tRNAs in immature sperm (Fig. S7F) are cleaved as sperm mature in the epididymis.

We next turned to potential functions of diet-regulated tRFs, using an embryonic stem (ES) cell system amenable to mechanistic analysis. Here, we interfered with the function of specific tRFs using antisense LNA-containing oligonucleotides, and assayed mRNA abundance as a readout of tRF inhibition. Most antisense oligos had no effect on mRNA abundance (Table S4), suggesting that the targeted tRFs are not functional in ES cells, or exert regulatory effects that are not assayed by mRNA abundance [START_REF] Sobala | Transfer RNA-derived fragments: origins, processing, and functions[END_REF]. In contrast, interfering with tRF-Gly-GCC resulted in dramatic upregulation of ~70 genes (Fig. 4A-C). These genes were unaffected by antisense oligos against other tRNA-Gly isoacceptors or against the middle or 3' end of tRNA-Gly-GCC (Fig. 4B). Curiously, the genes derepressed upon tRF-Gly-GCC inhibition are highly expressed in preimplantation embryos, and are regulated by the long terminal repeat (LTR) of the endogenous retroelement MERVL( 17 ) (Fig. 4D). tRF-Gly-GCC regulation of MERVL LTR-driven transcription could be recapitulated in LTR reporter cell lines, and is not secondary to translational effects of tRF-Gly-GCC inhibition (Figs.S8,S9,Table S5). Microinjection of antisense oligos targeting tRF-Gly-GCC into zygotes also resulted in derepression of MERVL targets later in development (Fig. 4E-F,Table S6), indicating that tRF-Gly-GCC also regulates MERVL in a more physiological context.

Given the robust connection between a diet-regulated small RNA and a highly specific set of target genes, we asked whether tRF-Gly-GCC targets are affected in preimplantation embryos generated using sperm from animals consuming Control or Low Protein diet. Single-embryo RNA-Seq ( 18 , 19 ) of individual mouse embryos cultured to various stages of development robustly clustered embryos by developmental stage (Figs. 5A-B,S10,Table S7), with the first two principal components of the dataset representing oocyte-derived transcripts (PC1), and the products of embryonic genome activation (PC2).

As single embryo RNA-Seq data are not suitable for identification of modest changes in individual mRNAs, we searched for consistent changes in larger genesets: the subset of MERVL targets that respond to tRF-Gly-GCC inhibition (Fig. 4), and remaining MERVL targets [START_REF] Macfarlan | Embryonic stem cell potency fluctuates with endogenous retrovirus activity[END_REF]. At the 2-cell stage both tRF-Gly-GCC targets and remaining MERVL targets were downregulated in Low Protein embryos relative to Control (Fig. 5C), consistent with the hypothesis that tRF-Gly-GCC in sperm affects expression of MERVL targets in early embryos. We carried out several independent tests of this hypothesis. First, we injected <40nt RNA populations purified from Control and Low Protein sperm into control zygotes, finding that Low Protein RNAs could inhibit tRF-Gly-GCC targets in 2-cell embryos (Figs. 5D,S11A), indicating that paternal diet can affect preimplantation gene regulation via RNAs in sperm. Second, further defining the relevant RNA from Low Protein sperm, microinjection of a synthetic tRF-Gly-GCC oligo resulted in repression of MERVL target genes in 2-cell embryos (Figs. 5E,S11B). Finally, as tRFs in sperm are gained during epididymal transit (Fig. S3), we generated embryos via intracytoplasmic sperm injection (ICSI) using testicular spermatozoa or cauda sperm. Consistent with the higher levels of tRF-Gly-GCC in cauda sperm, embryos generated using cauda sperm expressed MERVL targets at lower levels than embryos generated using testicular sperm (Figs. 5F,S12). Together, these findings all support the hypothesis that tRF-Gly-GCC in sperm is capable of delaying or repressing MERVL target expression in 2-cell embryos.

Lastly, we note that tRF-Gly-GCC is one of several abundant RNAs regulated by Low Protein diet, and MERVL-driven genes are not the only diet-responsive genes in preimplantation embryos. Most notably, ribosomal protein genes (RPGs) were downregulated in Low Protein embryos, and, correspondingly, Low Protein embryos develop slower than Controls (Fig. S13) [START_REF] Mitchell | Paternal diet-induced obesity impairs embryo development and implantation in the mouse[END_REF]. It remains to be determined whether altered preimplantation growth kinetics, or regulation of MERVL targets, could be responsible for eventual metabolic consequences in offspring. However, as the MERVL program is linked to totipotency [START_REF] Macfarlan | Embryonic stem cell potency fluctuates with endogenous retrovirus activity[END_REF], we speculate that tRF-Gly-GCC repression of MERVL-regulated genes could affect placental size or function, causing downstream effects on metabolism secondary to altered placentation [START_REF] Rando | I'm Eating for Two: Parental Dietary Effects on Offspring Metabolism[END_REF].

Taken together, our data identify a role for paternal diet in regulating the sperm epigenome in mammals. We show 1) that paternal diet can influence offspring phenotype via information in sperm, 2) that diet alters the level of small RNAs, including tRNA fragments, throughout the male reproductive tract and in mature sperm, and 3) that tRNA fragments can regulate expression of transcripts driven by endogenous retroelements. Our data also illuminate temporal dynamics of small RNA biogenesis during post-testicular sperm maturation, and suggest that epididymosomes traffic RNAs from somatic cells of the epididymis to maturing gametes.

One sentence summary

Abundant tRNA fragments in mammalian sperm are gained during post-testicular maturation in the epididymis, and regulate expression of endogenous retroelements upon delivery to the embryo.

"In Science" summary

Paternal dietary conditions have been shown to influence metabolic phenotypes in offspring in mammals. Sharma et al show that paternal diet affects levels of small RNAs in mammalian sperm. Small RNAs such as tRNA fragments are gained as sperm exit the testis and transit the epididymis, suggesting a role for soma to germline communication in small RNA biogenesis in mammals. Functionally, a key diet-regulated tRNA fragment, tRF-Gly-GCC, was shown to repress genes linked to an endogenous retroelement active in the preimplantation embryo. These results illuminate surprising aspects of small RNA biogenesis and function in mammalian reproduction. (F) Effects of epididymal passage on embryonic gene regulation. Intact sperm isolated from rete testis (n=12), or cauda epididymis (n=9), were injected into control oocytes, and mRNA abundance was analyzed as above.

Fig. 1 .

 1 Fig.1. Dietary effects on small RNAs in sperm (A) Size distribution of sequencing reads for cauda sperm small RNAs. (B-D) 5' tRNA fragments are shown schematically, with arrows indicating dominant 3' ends. (E) Dietary effects on sperm small RNA content. Scatterplot shows RNA abundance (ppm) for sperm isolated from Control (x axis, log 10 ) compared to Low Protein sperm (y axis), with various RNA classes indicated. Multiple points for tRFs result from sequence differences between genes encoding a given tRNA isoacceptor. (F) Heatmap showing RNAs responding to diet across eight paired sperm samples.

Fig. 2 .

 2 Fig.2. tRNA cleavage predominantly occurs in the epididymis (A) Sperm RNA payload diverges dramatically from testicular RNA. Scatterplot shows small RNAs in testis vs. sperm, as in Fig.1E. (B) Schematic of epididymis. Sperm exiting the testis enter the proximal (caput) epididymis, then proceed distally to corpus and cauda epididymis, and exit via the vas deferens. (C) tRFs are primarily generated in epididymis. Northern blots for 5' ends of tRNA-Gly-GCC or tRNA-Val-CAC on RNA extracted from testis, caput, and cauda epididymis. Arrow indicates ~30-34 nt 5' tRFs. 5S RNA serves as loading control. (D) Pie charts showing percentage of small RNAs mapping to the indicated features. (E) Scatterplot of small RNA abundance for sperm vs. epididymosomes. Sperm-enriched RNAs include piRNAs and fragments of mRNAs involved in spermatogenesis (eg Prm1), and represent RNAs synthesized during testicular spermatogenesis.

Fig. 3 .

 3 Fig.3. Changes in sperm tRF payload during epididymal transit (A) Proximal-distal biases observed for RNAs in epididymis are recapitulated in sperm samples. (B) Proximal-distal biases for tRFs, aggregated by anticodon, in epididymis and sperm. (C) TaqMan of the indicated tRFs in caput sperm and reconstituted sperm, showing gain of tRFs relative to let-7 (t-test p=0.05 for Gly-GCC, 0.004 for Val-CAC). (D) Deep sequencing of reconstituted sperm. tRFs are aggregated by codon, and normalized to levels of tRF-Glu-CTC. Caput vs. cauda differences were broadly recapitulated in reconstitutions, with tRFs such as tRF-Val-CAC being delivered to caput sperm via fusion with cauda epididymosomes.

Fig. 4 .

 4 Fig.4. tRF-Gly-GCC regulates MERVL-driven transcripts (A) Scatterplot shows mRNA abundance in ESCs transfected with GFP siRNA vs. LNA antisense to 5' end of tRF-Gly-GCC. (B) Effect of tRF-Gly-GCC inhibition on MERVL-regulated genes is isoacceptor-specific. Affymetrix data for ESCs transfected with LNA antisense oligos, relative to matched GFP controls, showing genes changing 2-fold in 2 or more samples. (C) RNA-Seq data for ESCs transfected with GFP siRNA, or anti-tRF-Gly-GCC. (D) Genomic context of tRF-Gly-GCC target genes, showing nearby MERVL LTRs. (E) tRF-Gly-GCC inhibition affects MERVL targets in embryos. Averaged single embryo RNA-Seq data for control (n=28) or tRF-inhibited (n=27) 4-cell stage embryos. Among 2fold upregulated genes, known MERVL targets( 17 ) are indicated. (F) Single embryo data for two MERVL targets.

Fig. 5 .

 5 Fig.5. Paternal dietary effects on preimplantation development (A) Embryos generated by IVF were cultured for varying times, then subject to single embryo RNA-Seq. (B) Single-embryo data for preimplantation embryos represented via PCA: first two principal components explain 74% of dataset variance. (C) mRNA abundance in 2-cell embryos generated via IVF using Control vs. Low Protein sperm (n=41 C and 39 LP). Cumulative distribution plots for tRF-Gly-GCC targets (p=4.5×10 -7 , KS test), other MERVL targets ( 17 ) (p=2.5×10 -13 ), and all remaining genes, showing percentage of genes with the average Log 2 (LP/C) indicated on the x axis. Low Protein embryos exhibit a significant shift to lower expression of MERVL targets. Bottom panels show individual embryo data for two targets. (D) Small RNAs isolated from Control or Low Protein cauda sperm were microinjected into control zygotes. RNA-Seq (n=42 C and 46 LP embryos) reveals downregulation of tRF-Gly-GCC targets (p=4.8×10 -14 ) driven by Low Protein RNAs. (E) Effects of synthetic tRF-Gly-GCC on 2-cell gene regulation, showing significant (p=0.0001) downregulation of target genes in embryos injected with tRF-Gly-GCC (n=26) vs. GFP controls (n=11). Inset shows effects of tRF-Glu-CTC (n=6).(F) Effects of epididymal passage on embryonic gene regulation. Intact sperm isolated from rete testis (n=12), or cauda epididymis (n=9), were injected into control oocytes, and mRNA abundance was analyzed as above.

Science. Author manuscript; available in PMC 2016 June 01.

ACKNOWLEDGEMENTS

We thank H.Florman, P.Visconti, R.Sadeh, N.Friedman for reading the manuscript, P.Visconti, M.Jungnickel for training in caput sperm purification, K.Sutton for training in ICSI, A.Shalek, A.Regev for training in single cell RNA-Seq, T.Fazzio, S.Hainer, B.Chen for assistance with ES studies, and J.Lawrence, K.Creamer for help with microscopy. Research was supported by NIH grants R01HD080224, DP1ES025458, March of Dimes grant FY13-1268. CCC was supported by a Helen Hay Whitney Postdoctoral Fellowship, AB by a Human Frontiers Science Program Fellowship. Data can be accessed at GEO, accessions GSE74537,GSE75613,GSE75622.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.