Virginie Vauthier

Sylvie Jaillard

Hubert Journel

Christèle Dubourg

Ralf Jockers
email: ralf.jockers@inserm.fr

Julie Dam

HOMOZYGOUS DELETION OF A 80 KB REGION COMPRISING THE DNAJ6C GENE AND PART OF THE LEPR GENE ON CHROMOSOME 1P31.3 IS ASSOCIATED WITH EARLY ONSET OBESITY, MENTAL RETARDATION AND EPILEPSY

Keywords: leptin receptor, OB-RGRP/endospanin-1, epilepsy, obesity, auxilin-1

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Introduction

Obesity is one of the greatest current public health challenges, not only in industrialized countries but also in developing countries [START_REF] Bonow | Diet, obesity, and cardiovascular risk[END_REF]. The obesity epidemic is driven by recent lifestyle and environmental changes together with the genetic heritage of each individual. Leptin has been identified as a key hormone of body weight regulation and energy homeostasis that acts on leptin receptors (OB-R) that are primarily expressed in the hypothalamus but also in other central and peripheral regions [START_REF] Myers | Obesity and leptin resistance: distinguishing cause from effect[END_REF]. The central role of leptin and its receptor is illustrated by the obese phenotype of rodents and humans carrying nonfunctional mutations in the ob and LEPR genes encoding leptin and its receptor, respectively [START_REF] Zhang | Positional cloning of the mouse obese gene and its human homologue[END_REF][START_REF] Tartaglia | Identification and expression cloning of a leptin receptor[END_REF]. To date, only 10 families have been reported with mutations in the LEPR gene [START_REF] Clement | A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction[END_REF][START_REF] Farooqi | Clinical and molecular genetic spectrum of congenital deficiency of the leptin receptor[END_REF][START_REF] Mazen | Homozygosity for a novel missense mutation in the leptin receptor gene (P316T) in two Egyptian cousins with severe early onset obesity[END_REF]. Homozygous carriers of these mutations show early onset obesity, hyperphagia, altered immune function and hypogonadotropic hypergonadism with the lack of pubertal growth spurt.

The genomic organization of the LEPR gene is complex. The P1 promoter of the LEPR gene generates two distinct transcripts through alternative splicing, the OB-R transcript and the Leptin Receptor Overlapping Transcript (LEPROT) that share the same 5' untranslated region but use different AUG initiation codons giving rise to two distinct open reading frames (Fig. 1A) [START_REF] Bailleul | The leptin receptor promoter controls expression of a second distinct protein[END_REF][START_REF] Seron | Endospanins regulate a post-internalization step of leptin receptor endocytotic pathway[END_REF]. The OB-R transcript encodes different OB-R protein isoforms including the signaling-competent long OB-Rb isoform. The LEPROT transcript encodes OB-R gene-related protein (OB-RGRP) or also called endospanin-1, which has no sequence homology with OB-R [START_REF] Bailleul | The leptin receptor promoter controls expression of a second distinct protein[END_REF]. Furthermore, OB-R/B219, a third transcript, is expressed from a second, internal, P2 promoter (Fig. 1A). This transcript generates different short OB-R isoforms (B219), with an expression pattern that is different from the receptors originating from the OB-R transcript (giving rise to long and short isoforms). Despite earlier suggestions that OB-R isoforms generated by the OB-R/B219 transcript might be involved in hematopoiesis [START_REF] Cioffi | Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction[END_REF], their function remains still largely unknown.

Recently it has been shown that Endospanin-1 regulates the number of OB-R at the cell surface in vitro by retaining the receptor in intracellular compartments through an interaction with OB-R [START_REF] Seron | Endospanins regulate a post-internalization step of leptin receptor endocytotic pathway[END_REF][START_REF] Couturier | Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity[END_REF]. The phenotype of knockout mice for endospanin-1 has not been described so far. Interestingly, shRNAmediated silencing of endospanin-1 specifically in the hypothalamic arcuate nucleus improves OB-Rdependent signaling and prevents against the development of diet-induced obesity [START_REF] Couturier | Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity[END_REF].

We describe here the phenotype of a 7-year-old boy carrying a homozygous deletion of a 80 kb region at chromosomal 1p31.3 region that includes most of the coding exons of DNAJ6C gene and the 5' part of the LEPR gene resulting in the abolishment of auxilin-1, OB-R and LEPROT transcripts but the maintenance of the OB-R/B219 transcript.

Case History And Clinical Examination

In 2004, a 3 year old male patient with early severe obesity was referred to the Genetic Counseling Unit, Vannes, France, to investigate his obesity-associated hyperphagia and developmental delay (fine motor problems associated with language learning difficulties). By 7 years old, he was 122cm tall and weighed 41 kg, with a BMI of 27.5 kg/m 2 thus above the 99th percentile defining a state of obesity (Fig. 1B-D).

The patient was born with a birth weight of 4090g, which is at the upper limit of normal birth weight (Fig. 1B). Starting from the age of 1, the BMI of the patient was higher than normal, mainly due to increased body weight (Fig. 1C) associated with elevated food-seeking behavior.

Clinical examinations of the patient (III1) showed normal height evolution until the age of 11 years. He exhibited mild dysmorphic features (round face, mild brachydactyly, undescended testis), obesity, mental retardation and epilepsy, but with no other clinical abnormalities. Past family history revealed that most members were overweight or obese (Fig. 1E, Table 1) and the patient's maternal grandmother (I2) as well as two paternal aunts (II1, II2) showed an epileptic history. The two latter (II1, II2) exhibited also mild mental retardation. However, in the present study, we were unable to further explore individuals from the paternal family.

The patient showed normal levels of serum lipids and hormones (Table 2). The number of blood cells was normal with the exception of low CD4+ T lymphocytes and increased CD8+ T lymphocytes that results in a low CD4/CD8 T-cell ratio. The number NK lymphocytes was higher than normal (Fig. 2A). A summary of the features of mental retardation and the epileptic syndrome of III1 are presented in Supplementary table 1.

Materials and Methods

Blood parameters

Plasma levels of hormones and lipids were determined by standard methods and immunophenotyping by flow cytometry with a FACS Calibur (BECTON -DICKINSON).

Array-CGH

Oligonucleotide array-CGH was performed using the Agilent Human Genome CGH microarray 44K (Agilent Technologies, Santa Clara, CA, USA). These microarrays comprise more than 44,000 60-mer oligonucleotide probes that span both coding and non-coding regions. Microarrays were scanned using the Agilent scanner G2565BA. Data were analyzed with Agilent DNA Analytics software to identify chromosome aberrations. Identification of probes with a significant gain or loss was based on the log 2 ratio plot deviation from 0 with cut-off values of 0.5 to 1 or -0.5 to -1 respectively. A value of -4 characterise a homozygous deletion.

Multiplex PCR/Liquid chromatography (MP-LC)

Duplex PCR was performed, associating unlabeled primers for an endogenous control gene, HMBS, and for OB-R/LEPR showing imbalance in the patient. Duplex for normal control, patient and parents gDNAs was performed as described elsewhere [START_REF] Bendavid | MLPA screening reveals novel subtelomeric rearrangements in holoprosencephaly[END_REF][START_REF] Jaillard | 2q23.1 microdeletion identified by array comparative genomic hybridisation: an emerging phenotype with Angelman-like features?[END_REF]. Data analysis was performed using the Navigator™ Software (Transgenomic, Omaha, NE, USA), normalisation was achieved with HMBS peak and relative peak intensities directly reflected OB-R/LEPR genomic copy number.

DNA extraction, RNA extraction and semi quantitative RT-PCR

DNA and RNA extractions and RT-PCR experiments were performed according to standard procedures (for primers see supplementary table 2).

Results

Blood chromosome analysis showed that the subject (III1) had a normal karyotype. Initial genetic tests excluded X Fragile Syndrome, the most common inherited cause of intellectual disability associated in some cases with obesity in males. Genetic tests on paternal chromosome 15q11-q13 as well as III1 clinical features (no hypotonia, no genital hypoplasia, no short stature) excluded Prader-Willi syndrome which is a complex genetic condition also characterized with obesity and intellectual impairment (data not shown). Array-CGH analysis revealed a chromosome 1 interstitial homozygous deletion on 1p31.3, confirmed by dye-swap (Fig. 1F) and excluded any other micro-deletion in the genome. The distal breakpoint mapped within the chromosome interval 65.604 Mb (the last non deleted probe) and 65.627 Mb (deletion starting point) from 1p telomere and the proximal one within the chromosome interval 65.707 (deletion ending point) and 65.744 Mb (first non deleted probe). Hence the deletion encompasses about 80 kilobases. To confirm array data, multiplex PCR/Liquid Chromatography (MPLC) experiments were performed for III1 and his parents. The homozygous deletion was confirmed in III1, inherited from asymptomatic and probably consanguineous parents, as each were heterozygous for the same deletion (Fig. 1G). The 80 kilobases deletion comprises only two known genes: (i) the DNAJ6C gene, encoding auxilin-1, a protein predominantly expressed in the brain that is required for clathrin recycling in cells [START_REF] Lemmon | Clathrin uncoating: Auxilin comes to life[END_REF], and (ii) the LEPR gene, known to encode the leptin receptor (OB-R) [START_REF] Tartaglia | The leptin receptor[END_REF] and endospanin-1 [START_REF] Bailleul | The leptin receptor promoter controls expression of a second distinct protein[END_REF][START_REF] Seron | Endospanins regulate a post-internalization step of leptin receptor endocytotic pathway[END_REF]. The deletion encompasses the 3' region of the DNAJ6C gene starting from DNAJ6C exon 5 and includes the 5' part of the LEPR gene comprising the proximal P1 promoter region and exons 1 and 2 (Fig. 1A,1F). MPLC analysis showed that both parents are heterozygous for the 1p31.3 deletion (Fig. 1G) and that 6 other related family members exhibited the same 1p31.3 deletion (Fig. 1E,1G).

To further confirm the absence of the 1p31.3 region in III1, specific PCR primers were designed to amplify this region in III1 and other family members (Fig. 1A, Supplementary Fig. 1A). As expected, primers specific for DNAJ6C gene exon 9 and for LEPR gene exon 2 readily revealed the expected amplicon in heterozygous family members but not in III1.

The deletion of 91% of DNA6JC coding region is in accordance with the absence of functional auxilin-1 transcript in III1 as compared to wild type or heterozygous carriers (Fig. 2B).

The presence of LEPR gene exon 8 was detected in all cases consistent with the partial deletion of the LEPR gene. The corresponding mRNAs for auxilin-1, LEPROT and OB-R were absent in blood samples of III1 but present in heterozygous family members (Fig. 2B). Similar results were obtained in skin mRNA preparations (Supplementary Fig. 1B).

The presence of OB-R/B219 transcript generated from the internal P2 promoter was confirmed in mRNA samples prepared from III1 blood cells (Fig. 2B). Expression in blood cells but not in skin cells (data not shown) is in accordance with previous reports [START_REF] Bailleul | The leptin receptor promoter controls expression of a second distinct protein[END_REF][START_REF] Cioffi | Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction[END_REF].

Endospanin-1 has a unique homologue in the human genome called endospanin-2 that is encoded by the LEPROTL1 gene located on chromosome 8p21.1-8p21.2 [START_REF] Seron | Endospanins regulate a post-internalization step of leptin receptor endocytotic pathway[END_REF][START_REF] Huang | Cloning and characterization of a novel human leptin receptor overlapping transcript-like 1 gene (LEPROTL1)[END_REF]. As recent studies in transfected cells suggest that both endospanins negatively regulate OB-R surface expression, a compensatory effect of one endospanin in the absence of the other might be possible. However, semi-quantitative RT-PCR analysis showed that similar amounts of endospanin-2 mRNA were present in blood samples of III1, heterozygous carriers and wild-type family members (Supplementary Fig. 1C, Fig. 2C).

Discussion

In the present study, we report for the first time a patient (III1) with a homozygous deletion within the chromosomal 1p31.3 region comprising only two predicted genes, DNAJ6C and LEPR genes. Whereas III1 with the homozygous deletion showed early on-set obesity, mental retardation and epilepsy, 8 heterozygous carriers including a sibling of III1 are also overweight or obese, but do not exhibit mental retardation or epilepsy (except 1 member).

DNAJ6C encodes auxilin-1, a protein expressed in the brain and acting as a co-chaperone to support Hsc70-dependent clathrin uncoating of clathrin-coated vesicles in neurons [START_REF] Lemmon | Clathrin uncoating: Auxilin comes to life[END_REF]. Mice with targeted deletion of auxilin-1 show recycling and endocytosis defects of synaptic vesicles and are characterized by early postnatal mortality likely due to neurological and cognitive impairment [START_REF] Yim | Endocytosis and clathrin-uncoating defects at synapses of auxilin knockout mice[END_REF]. Recently, major reports demonstrated that, in mice and humans, deficiency of genes involved in synaptic vesicle recycling and endocytosis lead to impairment of neurocognitive functions characterized by mental retardation, defect in learning/memory and seizures: endophilin (18), oligophrenin-1 (19-21), dynamin-1 [START_REF] Boumil | A missense mutation in a highly conserved alternate exon of dynamin-1 causes epilepsy in fitful mice[END_REF], amphiphysin-1 [START_REF] Paolo | Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice[END_REF], synaptojanin-1 [START_REF] Cremona | Essential role of phosphoinositide metabolism in synaptic vesicle recycling[END_REF][START_REF] Kim | Delayed reentry of recycling vesicles into the fusion-competent synaptic vesicle pool in synaptojanin 1 knockout mice[END_REF]. Importantly, given the widespread impact of synaptic neurotransmission on brain functions, many of these deficiencies led to perinatal mortality due to severe neurologic defects [START_REF] Yim | Endocytosis and clathrin-uncoating defects at synapses of auxilin knockout mice[END_REF][START_REF] Milosevic | Recruitment of endophilin to clathrincoated pit necks is required for efficient vesicle uncoating after fission[END_REF][START_REF] Paolo | Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice[END_REF][START_REF] Cremona | Essential role of phosphoinositide metabolism in synaptic vesicle recycling[END_REF][START_REF] Kim | Delayed reentry of recycling vesicles into the fusion-competent synaptic vesicle pool in synaptojanin 1 knockout mice[END_REF]. We propose that auxilin-1 could be a new potential candidate gene for mental retardation and/or seizure, which requires further investigation in other human genetic studies.

Deletion of exons 1-2 and of P1 promoter of LEPR gene eliminates OB-R and LEPROT transcripts but not the OB-R/B219 transcript, which uses the internal P2 promoter. The phenotype of previously reported patients with non-functional OB-R resembles that of III1 described in our study: early onset obesity, hyperphagia and modification of the immune cells. Moreover, the high circulating leptin level of 50 ng/mL in the patient (normal range for boys of similar age is below 11.2 ng/mL), may explained the absence of a putative negative feedback loop between leptin signaling and leptin secretion. Consistently, decreased CD4/CD8 T lymphocyte ratios have also been observed in humans with congenital leptin deficiency [START_REF] Farooqi | Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency[END_REF]. The lack of pubertal growth spurt and hypogonadotropic hypergonadism reported for patients with leptin or OB-R deficiency has not yet been observed, as III1 is too young. Thus, obesity and defects in immune cells observed in III1 are most likely explained by the absence of the signalingcompetent long OB-Rb isoform. Mental retardation and epilepsy are unlikely to be causally related to the absence of OB-Rb as other reported mutations of the leptin pathway do not present this phenotype.

Whereas previously described mutations of the LEPR gene concern all OB-R and OB-R/B219 transcripts equally, deletion of LEPR gene exons 1 and 2, identified in our study, only abolishes OB-Ra/b transcript leaving the OB-R/B219 transcripts fully functional. Interestingly, the obese phenotype and the modified CD4+/CD8+ T-cell ratio observed in III1 supports the idea that OB-R/B219 transcripts are not likely to be involved in these functions. Some complex pleiotropic syndromes in which obesity is only one of the clinical phenotype of developmental anomalies are largely described [START_REF] O'rahilly | Minireview: human obesity-lessons from monogenic disorders[END_REF]. Many of the clinical phenotypes characterizing these syndromes were not seen in III1 and his family. Moreover, genetic analysis did not confirm the presence of X Fragile syndrome and Prader-Willi syndrome in III1. Human deficiency of BDNF [START_REF] Gray | Hyperphagia, severe obesity, impaired cognitive function, and hyperactivity associated with functional loss of one copy of the brain-derived neurotrophic factor (BDNF) gene[END_REF] or of its cognate receptor TrkB [START_REF] Yeo | A de novo mutation affecting human TrkB associated with severe obesity and developmental delay[END_REF] was also shown to trigger severe obesity, intellectual abilities and seizures. However, III1 does not likely have such mutations since BDNF or TrkB subjects also developed hyperactivity, impairment of nociception, hypotonia which were not observed in III1. This is the first study reporting the homozygous deletion of the LEPROT transcript of the LEPR gene. No other mutations have been reported so far for this transcript in humans. Endospanin-1, the corresponding protein of the LEPROT transcript, has been recently shown to regulate OB-R function [START_REF] Seron | Endospanins regulate a post-internalization step of leptin receptor endocytotic pathway[END_REF][START_REF] Couturier | Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity[END_REF]. However, due to the simultaneous absence of the signaling-competent OB-Rb isoform in III1, we are unable to confirm this prediction. Normal IGF1 levels and normal growth of III1 also excludes any OB-Rindependent effects on these parameters as suspected from a transgenic mouse model over-expressing endospanin-1 [START_REF] Touvier | LEPROT and LEPROTL1 cooperatively decrease hepatic growth hormone action in mice[END_REF]. Our study allows us to postulate a preliminary phenotype of endospanin-1 deficiency in humans. As the phenotype of III1 is likely to be dominated by the absence of auxilin-1 (mental retardation, epilepsy) and OB-Rb (early on-set obesity, modified immune cell composition), it is likely that the absence of endospanin-1 has no major phenotypic consequences in humans in the context of OB-Rb deficiency. Co-evolution of both transcripts under the control of the same promoter further supports this conclusion.

In conclusion, we describe here a unique homozygous 80 kb deletion in the chromosomal 1p31.3 region that confirms the early onset obesity of individuals with OB-R deficiency and suggests for the first time the phenotype of auxilin-1 and endospanin-1 deficiency in humans.

Acknowledgements: This work was supported by grants from the FP7 "EUROCHIP", Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Direction de l'Hospitalisation et de l'Organisation des Soins (DHOS) and a research fellowship of the Fondation pour la Recherche Médicale (to VV). We thank Drs Y. Cano, A. Bouvet, P Pouedras and teams from the "Service de Pédiatrie" (Centre Hospitalier Bretagne Atlantique,), Dr V. Gauthier (CHU, Nantes), Pr J. Mosser (Biogenouest transcriptomic platform, Rennes), K. Kerouredan (Centre d'action médico-sociale précoce (CAMSP), Vannes) for help with the family studies. We gratefully thank the subject and his family for their consent and participation. Contributions: V.V. and S.J. performed research and analyzed data; R.J. and J.D. wrote the manuscript; V.V., C.D. and H.J. reviewed/edited the manuscript and contributed to discussion; R.J., H.J., J.D. and C.D. managed the project, H.J. performed medical examinations.

Conflict-of-interest disclosure:

The authors declare no competing financial interests.

Figure 1 .

 1 Figure 1. Deletion of chromosomal 1p31.3 region comprising the DNAJ6C and LEPR gene. A.

Figure 2 :

 2 Figure 2: Characterization of gene expression in 5 family members. A.T, B and NK cell population

Table 1 :

 1 General phenotype of the family members including age, genotype, height, body weight (BW), BMI and other diseases. values § to be compared to normal range of childhood BMI which are agedependant in children (see Fig.1D).

Table 2 :

 2 Metabolic and endocrine features of 4 family members including the patient (III1) and its 2 sisters. Age and sex-specific normal ranges for leptin 2.4-11.2 ng/mL for 3-11 years old, <14.8 for women, <7.4 for men; for estradiol, < 0.076 pmol/L for clinically prepubertal persons, 0.01-0.75 for women in follicular phase; for testosterone, <1.43 ng/mL for clinically prepubertal persons; for Insulinlike Growth factor 1 (IGF-1), 6-39 for < 10 years old, 15-130 for 10-13 years old, 10-51 for > 13 years old; for Follicle-Stimulating hormone (FSH), <1U/L for clinically prepubertal subjects, 1-18 for pubertal subjects; for Luteiizing Hormone (LH) < 1U/L for clinically prepubertal subjects, 1.5-13 for pubertal subjects. * indicates out of normal range. Supplementary figure 1: A. PCR on genomic DNA extracted from blood cells. B. Semi-quantitative RT-PCR from mRNA extracted from skin biopsies. C. Semi-quantitative RT-PCR from mRNA extracted from blood cells

Primers Sequences Gene

Genomic


J Maysonnave

N Jukam

M S M Ibrahim

K Maussang

J Madéo

P Cavalié

P Dean

S P Khanna

D P Steenson

E H Linfield

A G Davies

J Tignon

S S Dhillon

An integrated injection seeded THz source and amplifier for time-domain spectroscopy

A terahertz (THz) frequency quantum cascade laser (QCL) is used as an integrated injection seeded source and amplifier for use in THz time-domain spectroscopy. A THz input pulse is generated inside a QCL, by illuminating the laser facet with a near-infrared pulse from a femtosecond laser, and amplified using gain switching. The THz output from the QCL is found to saturate upon increasing the amplitude of the THz input power, which indicates that the QCL is operating in an injection seeded regime.

In the technique of terahertz (THz) frequency timedomain spectroscopy (TDS), THz radiation is generated and detected using femtosecond (fs) near-infrared laser pulses, enabling measurement of both the THz amplitude and phase [1]. In TDS systems, the THz radiation can be generated when the near-infrared fs laser illuminates a GaAs photo-conductive antenna [2], travels through an electro-optic crystal with a non-vanishing χ χ χ χ 2 [3], or is focused in air [4]. In all these cases, the THz field amplitude scales linearly with the power of the fs pulse, until saturation takes place. One strategy to increase further THz field amplitudes is to use higher fs pulse energies, and TDS systems that utilize mJ pulse energies are becoming increasingly common [5]. Although this is a promising laboratory-based technique, it is not practical for applications outside the laboratory owing to the large cost and size of regenerative amplified laser systems.

An alternative approach to generate higher THz fields is to amplify THz waves directly after generation, and hence enable low-power fs laser pulses to generate high THz wave amplitudes. THz frequency quantum cascade lasers (QCLs) are natural candidates for THz amplifiers, and have already been used as external THz amplifiers to measure the QCL gain. [6][7][8]. In the steady-state, external amplification is limited by gain clamping and depends on the facet reflectivity [9]. Gain switching with RF pulses can be used to unclamp the gain in the QCL and augment external pulse amplification [10]. Amplifying THz 'seed' pulses in QCLs with sufficiently long RF pulses [11] can lead to coherent injection seeding where the QCL emission is phase-locked to the fs laser. This enables phase-resolved detection of the QCL emission in the timedomain, and permits the use of QCLs as intense THz sources for TDS. However, if the THz seed pulses are generated by external photoconductive antennas, the THz must be coupled from free space into the QCL cavity. This introduces large coupling losses that are associated impedance mismatch between the cavity and free space and can negate the effect of THz amplification.

In this letter, injection seeding of a QCL is achieved with THz seed pulses that are generated directly inside the QCL cavity. This results in a completely integrated QCLbased source for TDS. Specifically the THz seed pulses are generated inside the QCL cavity [12] by illuminating one of the facets with a near-infrared fs laser beam (Fig 1a). This removes the need to use a photoconductive THz emitter to generate the THz seed pulses and parabolic mirrors to couple the THz seed pulses into the cavity (Fig 1b). Gain switching of the QCL is used to achieve large amplification of the internally generated THz seed pulses. A coupled cavity scheme allows the amplitude of the input seed pulse to be increased without affecting the QCL gain [13,14]. We find that for sufficient THz seed amplitudes, the amplified seed pulses saturate the QCL gain, which indicates that injection seeding takes place via integrated seed generation.

To enter the injection seeding regime, the amplitude of the THz pulse generated at the facet must be sufficiently large to prevent the QCL from being 'seeded' by amplified spontaneous emission. Since the THz pulse amplitude generated at the facet increases with applied voltage [12], it is therefore desirable to apply a large voltage across the facet. However, QCL operation ceases once the applied bias becomes too large owing to misalignment of the injector and upper level laser state. In order to decouple the facet voltage from the QCL bias voltage, focused ion beam (FIB) etching was used to remove a 20 µm wide portion of the gold contact and the heavily-doped n-GaAs contact layer on the top of the laser ridge. This splits the QCL cavity into a short 305 µm seeding section used as the THz pulse generator, and a long 2.5 mm QCL gain section for THz pulse amplification. The resistance between these two sections (~35 Ω) is several times larger than the QCL differential resistance (3-6 Ω). Although, the two sections are not completely electrically isolated; removing the top contact in this way eliminates the need to etch through the entire active region using reactive ion etching [13,14]. As shown in Fig. 1d, independently biasing the seeding section decreases the QCL output power. This is most likely due to the leakage current between the seeding and QCL section and increased THz losses in the seeding section. The QCL is based on a bound-to-continuum design, and processed into a single plasmon waveguide with a lateral dimension of 180 µm and a total cavity length of 2.85 mm. The QCL is soldered to a gold-plated copper mount and placed in a continuous flow cryostat. Measurements are taken at 10 K, and laser emission is centered at 2.5 THz.

Before the arrival of the fs pulse (80fs, 808nm, 6nJ) that generates a THz pulse in the seeding section, the gain section is biased below threshold. When the THz pulse is generated in the QCL, an RF bias pulse (triggered by the fs laser) is applied to the gain section. This places the QCL in a transient state where the gain is much greater than the clamped gain, and permits large amplification [10]. Fig. 1c shows a schematic diagram of the electronic arrangement. A fast photodiode generates the RF pulses when illuminated with the fs laser. The RF pulses are amplified by a pre-amplifier that is operated in the saturation regime to produce RF pulses with a relatively flat top. The RF pulses are subsequently amplified by a power amplifier and applied to the QCL. The power of the incident RF pulses (Fig. 1e) is ~6 W, and about 40% of the RF power is injected into the QCL. Faster electronics permit shorter RF rise times and pulse durations compared with studies of external injection seeding [11]. A bias tee also allows the QCL gain section to be biased with a quasi-DC voltage (10 kHz), which reduces the RF power necessary to drive the QCL above threshold. The quasi-DC bias is set to ensure that the RF pulses drive the QCL to the bias at which maximum gain occurs (i.e. just before misalignment of the QCL band-structure). The seeding section is biased at 3.8V (~180A/cm 2 ) which roughly corresponds with Jmax in Fig. 1d. The RF pulses and QCL gain section are modulated at 10kHz (25% duty cycle) while the seeding section is modulated at 20 kHz i.e. at twice the duty cycle of the QCL gain section and RF pulses modulation. This permits lock-in detection of the difference field, which removes the unamplified low frequency components of the THz signal. Fig. 2 shows the QCL output when the RF drives the QCL above threshold. After the initial THz pulse is generated in the seeding section, it traverses the laser cavity and experiences gain (near the laser frequency) and losses (at other frequencies). At the opposite facet, part of the THz pulse is reflected back into the cavity and part is transmitted out of the QCL. This process will then continue with multiple passes traveling through the cavity. The output of the QCL thus consists of several THz pulses separated by the round-trip time (~71 ps) of the cavity. A pair of parabolic mirrors collimates and focuses the transmitted THz pulses onto a 200-µm-thick <110> ZnTe crystal for electro-optic detection.

As shown in Fig. 2a, the THz pulses undergo large amplification when the RF pulse brings the QCL bias above threshold and unclamps the gain. Figs. 2b and2c are zoomed in portions of the 1st and 4th THz pulses in Fig 2a, that show the phase-resolved nature of the measurement in Fig. 2a. After three round-trips the difference field is amplified by more than a factor of 80, and the output field is almost two orders of magnitude larger than the input field. The amplified THz emission lasts for approximately the same time as the RF pulse duration in Fig. 1e. Since there is no significant reflection between the seeding section and the gain section, no secondary THz seed pulses are produced. Although later THz pulses undergo dispersion, the THz pulses remain ) RF pulses are created with a fast photodiode (PD), and amplified. An electronic delay line is used to vary the RF pulse arrival time. The RF pulse train is modulated with a 50 Ω switch, and the pulse amplitude can be varied with a variable attenuator. A bias tee allows a quasi-DC voltage to be applied to the QCL along with the RF. A 20dB pickoff tee (RF probe) allows the RF shape to be monitored. d) d) d) d) Lightcurrent density (V-J) plots for the device with the seeding section biased at 0V (black), 3.8V (~180A/cm 2 ) (red), and shorted to the QCL section (blue). e) e) e) e) The RF pulse sent to the QCL measured with the pick-off tee on an 8 GHz sampling oscilloscope (50Ω). distinct. The maximum peak-to-peak field (40 V/cm corresponding to an intensity of 1.8 W/cm 2 ) is significantly greater than studies of external injection seeding on a 2.1THz QCL [11], and is due to the higher output powers of the 2.5THz devices (~20mW) compared to the 2.1THz device (1.5mW). From the ratio of the spectral amplitudes of successive THz pulses in Fig. 2a, we can find the average difference between the unclamped gain and the clamped gain, which we define as the excess gain. A positive excess gain will result in a net amplification of a pulse that undergoes a round-trip through the QCL cavity. The largest value of the average excess gain (at the laser frequency) is 9 cm -1 and occurs between the 2 nd and 3 rd pulses in Fig. 2a.

To determine if the THz input pulses saturate the QCL gain (and are thus injection seeding the QCL), the output QCL field can be studied as function of the input THz pulse amplitude by varying the voltage applied to the seeding section. The peak-to-peak fields of the 1st, 5th and 6th output THz pulses are plotted in Fig. 3 as a function of the seeding section voltage. The 1st output THz pulse corresponds to a single pass through the cavity. Although the THz radiation generated at the facet cannot be directly measured, it is proportional to the amplitude of the 1st emitted THz pulse. The amplitude of this pulse increases almost linearly for seeding section voltage less than 3.5 V. For higher voltages, the input amplitude continues to increase with seeding section voltage although the slope decreases slightly. When the seeding section voltage (and hence the amplitude of the generated THz pulse) is small, the peak-to-peak field of the 5th and 6th pulses also increases with voltage. However, for large seeding voltages the peak amplitudes of the 5th and 6th pulses are flat even though the amplitude of the 1st pulse keeps increasing. This indicates that the gain of the laser is saturated (clamped) by the amplified THz pulses, and the laser emission has entered the injection seeded regime.

In conclusion, we have demonstrated an integrated QCL-based source of THz radiation suitable for TDS. THz pulses are generated directly inside a coupled-cavity QCL by illuminating the facet with fs near-infrared laser pulses, and are subsequently amplified to high field values using gain switching until injection seeding of the QCL is achieved. The THz signal generated in the seed element is thus increased by amplifying the THz wave using the high gain available from the QCL gain section, rather than by increasing the fs laser power. Decoupling the strength of the THz signal from the fs laser power could make TDS more practical by reducing the need for higher power fs lasers.

Figure 1

 1 Figure 1 a) a) a) a) THz pulses are generated inside the QCL by illuminating the seeding section with an fs laser. b) b) b) b) THz pulses created by an external photoconductive antenna are coupled into a QCL with parabolic mirrors. c) c) c) c) RF pulses are created with a fast photodiode (PD), and amplified. An electronic delay line is used to vary the RF pulse arrival time. The RF pulse train is modulated with a 50 Ω switch, and the pulse amplitude can be varied with a variable attenuator. A bias tee allows a quasi-DC voltage to be applied to the QCL along with the RF. A 20dB pickoff tee (RF probe) allows the RF shape to be monitored. d) d) d) d) Lightcurrent density (V-J) plots for the device with the seeding section biased at 0V (black), 3.8V (~180A/cm 2 ) (red), and shorted to the QCL section (blue). e) e) e) e) The RF pulse sent to the QCL measured with the pick-off tee on an 8 GHz sampling oscilloscope (50Ω).

Figure 2

 2 Figure 2 a) a) a) a) The THz difference field emitted from the QCL measured with electro-optic sampling. The incident RF pulse amplitude is 6 W and the quasi-DC bias is 62 A/cm 2 . b) b) b) b) Enlarged view of the 1st THz pulse that undergoes a single pass through the QCL. c) c) c) c) Enlarged view of the 4th THz pulse, which shows the phase-resolved nature of the measurement.

Figure 3

 3 Figure3Maximum peak-to-peak (p-p) fields of the 1st (black), fifth (green) and sixth (blue) THz pulses versus the voltage applied to the seeding facet. For values of the seeding voltage >4 V, the amplitudes of the fifth and sixth THz pulses saturate, while the amplitude of the 1st THz pulse continues to increase.

Acknowledgements

Acknowledgements Acknowledgements Acknowledgements This work was supported by ANR projects "HI-TEQ", "THINQE-PINQE", ERC Advanced Grant projects "NOTES" and "TOSCA", the EPSRC (UK), and the MARA Agency (Malaysia).

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