Katja Ritz

Barbera Van Schaik

Marja E Jakobs

Antoine H Van Kampen

Eleonora Aronica

Marina A Tijssen

PhD Frank Baas
email: f.baas@amc.uva.nl

Barbera Dc Van Schaik

SGCE isoform characterization and expression in human brain: implications for Myoclonus-Dystonia pathogenesis?

Keywords: Myoclonus-Dystonia, SGCE, deep sequencing, alternative splicing, imprinting, cerebellum

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Myoclonus-Dystonia (M-D) is a movement disorder characterized by myoclonic jerks and dystonic features, usually affecting the upper part of the body. Little is known about the pathological basis of M-D symptoms. Electrophysiological studies in man suggest that myoclonic symptoms are of subcortical origin. [START_REF] Klein | Myoclonus and myoclonus-dystonias[END_REF][START_REF] Nardocci | Myoclonus-dystonia syndrome: clinical presentation, disease course, and genetic features in 11 families[END_REF][START_REF] Marelli | A Neurophysiological Study of Myoclonus in Patients with DYT11 Myoclonus-Dystonia Syndrome[END_REF] In general, dystonia is thought to arise from dysfunction of the basal ganglia. [START_REF] Breakefield | The pathophysiological basis of dystonias[END_REF] About 50% of M-D patients that were classified as definite M-D carry a mutation in the widely expressed ε-sarcoglycan (SGCE). [START_REF] Zimprich | Mutations in the gene encoding epsilonsarcoglycan cause myoclonus-dystonia syndrome[END_REF][START_REF] Grunewald | Myoclonus-dystonia: significance of large SGCE deletions[END_REF][START_REF] Ritz | Myoclonus-dystonia: clinical and genetic evaluation of a large cohort[END_REF] The genetic cause in the remaining patients is still unclear. A second locus has been reported in one large M-D family (DYT15, 18p11), but no gene has been identified yet. 8;9 SGCE is part of the sarcoglycan family that consists of N-glycosylated transmembrane proteins. Six different sarcoglycans have been identified so far (α-, β-, γ-, δ-, ε-, ζ-) but little is known about the function of particular sarcoglycan members and their function in different tissues. [START_REF] Ozawa | Molecular and cell biology of the sarcoglycan complex[END_REF] In muscle, sarcoglycans form a heterotetrameric complex that is constituent of the dystrophin-associated protein complex. This complex mediates the structural stability of the plasma membrane and interactions between the extracellular matrix and the cytoskeleton. [START_REF] Waite | The neurobiology of the dystrophinassociated glycoprotein complex[END_REF] Mutations in other sarcoglycans, α-, β-, γ-and δsarcoglycan, lead to different forms of limb-girdle muscular dystrophy, characterized by progressive muscle weakness. [START_REF] Ozawa | Molecular and cell biology of the sarcoglycan complex[END_REF] Little is known about composition and function of the dystrophin-associated protein complex in brain. SGCE is highly homologous to αsarcoglycan, [START_REF] Mcnally | Human epsilon-sarcoglycan is highly related to alpha-sarcoglycan (adhalin), the limb girdle muscular dystrophy 2D gene[END_REF] but no muscle or myocardial muscle abnormalities have been identified in M-D patients. [START_REF] Hjermind | No muscle involvement in myoclonusdystonia caused by epsilon-sarcoglycan gene mutations[END_REF] Understanding the exclusively neurological phenotype of the disease is still a major challenge. SGCE is an imprinted gene 14;15 meaning that a loss of function mutation in the expressed allele will be dominant. All reported mutations behave as null alleles, since they result in premature stop codons and are predicted to lead to nonsense-mediated decay.

There is also evidence that SGCE missense mutations lead to loss of function. [START_REF] Esapa | SGCE missense mutations that cause myoclonusdystonia syndrome impair epsilon-sarcoglycan trafficking to the plasma membrane: modulation by ubiquitination and torsinA[END_REF] SGCE is widely expressed which suggests either redundancy for this protein in non-brain tissue or a brain-specific function for SGCE.

There are four known alternatively spliced exons in the SGCE gene (exon 2, 8, 10 and 11b, Fig. 1A) with exon 11b as a brain-specific exon. [START_REF] Nishiyama | Identification and characterization of epsilon-sarcoglycans in the central nervous system[END_REF] Recently, a new brain-specific alternatively spliced exon (exon 11c, an elongated exon 11b) has been identified in mice. [START_REF] Yokoi | Exclusive paternal expression and novel alternatively spliced variants of epsilon-sarcoglycan mRNA in mouse brain[END_REF] Transcripts containing either exon 11b or 11c encode proteins with a different C-terminal sequence containing a PDZ-binding motif. This motif is a protein-interaction domain and thus may contribute to a unique SGCE function in brain. The ubiquitous SGCE protein and the brain-specific protein (exon 11b) are localized in different synaptosomal membrane fractions: post-and pre-synaptic membrane, respectively. [START_REF] Nishiyama | Identification and characterization of epsilon-sarcoglycans in the central nervous system[END_REF] It has been speculated that both isoforms play different roles at neuronal synapses.

The aim of our study was to characterize the quantitative and qualitative expression pattern of SGCE isoforms in human brain in order to identify brain regions associated with M-D.

Methods

Sample collection and preparation

Human tissue was obtained from six control subjects without neurological symptoms (Table 1). In addition, two blood samples were obtained from control subjects. SGCE mutations were excluded by direct sequencing of the coding region. Human tissues were obtained from the Department of neuropathology of the Academic Medical Center (University of Amsterdam, The Netherlands) and informed consent was obtained for research purposes in all control subjects. Mouse (cerebral cortex), rat (cerebral cortex) and zebrafish (100 embryos, 24 hours after fertilization) tissue was obtained from wild type animals (approved by local committee). DNA extractions from frozen brain tissues and whole-blood samples were performed using standard procedures. Twenty to 40 sections (depending on tissue size, 20µm) were cut per sample and dissolved in TRIzol Reagent (Invitrogen, Breda, The Netherlands) for subsequent RNA isolation, or in SE buffer (750mM NaCl pH 8, 250mM EDTA, 1% SDS) containing Proteinase K for DNA isolation.

Total RNA was isolated with QIAcube instrument (Qiagen, Venlo, The Netherlands) using RNeasy Mini protocol or using the PAXgene system (Qiagen) for blood samples.

Methylation-sensitive high resolution melting assay

To determine the degree of methylation in the promotor region of SGCE we applied a methylation-sensitive high resolution melting (MS-HRM) assay. The region amplified (nucleotide position -1148 to -773 relative to the start ATG) contains 25 CpG sites and has been shown to be differentially methylated. [START_REF] Muller | Evidence that paternal expression of the epsilon-Sarcoglycan gene accounts for reduced penetrance in myoclonus-dystonia[END_REF] 1µg of genomic DNA was chemically modified with sodium bisulfite using the EZ Methylation kit (Zymo Research, Orange, CA, USA) and amplified in the presence of ResoLight HRM dye (Roche Diagnostics, Almere, The Netherlands). Both, methylated and unmethylated, strands were amplified. [START_REF] Muller | Evidence that paternal expression of the epsilon-Sarcoglycan gene accounts for reduced penetrance in myoclonus-dystonia[END_REF] MS-HRM analysis was performed with the LightCycler 480 (Roche Diagnostics) and analyzed with LightCycler 480 gene scanning software module. In each run a set of standards of the identical SGCE promotor region was included (100%, 50%, 25%, 12.5%, 6.25% and 0% methylated). Different brain regions of five control subjects (Subject 1, 2, 4, 5, 6), muscle tissue of two control subjects (Subject 2, 3) and blood of 12 definite M-D SGCE nonmutation carriers [START_REF] Ritz | Myoclonus-dystonia: clinical and genetic evaluation of a large cohort[END_REF] were tested. A subset of PCR amplified samples was cloned (pGEM-T Easy vector, Promega, Leiden, The Netherlands) and sequenced to verify results of the methylation assay (ABI big dye v3.1 chemistry, ABI 3730 sequencer, Applied Biosystems, Foster City, CA, USA).

Ultra deep amplicon sequencing

cDNA was synthesized from 1µg of total RNA with oligodT 12 -VN primers and 1µl cDNA was subjected to a 10µl PCR reaction. PCR reactions were performed using fusion primers consisting of a 19 bp fixed sequence (Roche/454 GS FLX, A or B sequence at the 5'end) and a target-specific sequence (3'end). We added a 5 nucleotide multiplex identifier-tag to allow for multiplexing of samples (primer sequences and PCR conditions are available on request). Amplicons were processed and ultra deep sequenced using the 454 GS FLX system (Roche Diagnostics) according to manufacturer's instructions.

Data analysis

In order to identify all alternatively spliced events, sequences were first grouped by a multiplex identifier-tag. Secondly, we used the BLAT algorithm (BLAST-like alignment tool) to compare each sequence with all other sequences to identify similar exon combinations per sample. Sequences were grouped when they encompassed at least 210 nt, had a percent identity of at least 98%, a score above 105 and a query coverage above 98%.

The resulting groups were mapped to the respective chromosome (BLAT algorithm, hg18 build) and analyzed in the UCSC genome browser and using CodonCode Aligner software 3.0.1 (Dedham, MA, USA).

Real-time quantitative PCR

1µl cDNA was amplified in triplicates using the LightCycler480 (Roche Diagnostics) in a final volume of 10µl containing SYBR Green I Master mix (Roche Diagnostics). Intron spanning primers were designed to target either all SGCE isoforms or the brain-specific isoform containing exon 11b. All quantitative PCR (qPCR) results were normalized against the housekeeping genes GAPDH and eEF-1 alpha. Data was analyzed using LinRegPCR analysis program. [START_REF] Ruijter | Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data[END_REF]

In situ hybridization

In situ hybridization (ISH) was performed as previously described. [START_REF] Budde | tRNA splicing endonuclease mutations cause pontocerebellar hypoplasia[END_REF] Two 5'-fluoresceinlabeled 19mer antisense oligonucleotides containing locked nucleic acid and 2'-O-methyl (2OME) RNA moieties (Ribotask ApS, Odense, Denmark) were designed: one targeting the brain-specific alternatively spliced exon 11b (5'-AacGaaAauCucCugTagT-3', locked nucleic acid residues are given in capital letters, 2'-O-methyl-RNA in lower case) and one detecting all isoforms targeting the constitutive SGCE exon 3 (5'-TagGacTccAucAcuAuaT-3'). Brain tissue was treated with Proteinase K (200µg/ml, Roche Diagnostics) at 50°C for 10 minutes. Probe annealing was performed at 53°C for 1.5 hours. Peroxidase activity was visualized using NovaRED (Vectorlabs, Burlingame, CA) and counterstained with hematoxylin. In an adult control subject we tested different human brain regions (cerebellum, mesencephalon, caudate nucleus, putamen, globus pallidus, cerebral cortex and hippocampus). Skeletal muscle, heart, kidney and liver sections were used to confirm specificity of the brain-specific probe. As a control for specific binding, brain material of a SGCE mutation carrier (c.835_839del, p.Lys280SerfsX16, 50 h post mortem) was analyzed. This tissue was obtained from the NICHD Brain and Tissue Bank (Baltimore, MD, USA).

Results

SGCE imprinting and M-D

SGCE is widely expressed and there is thus far no explanation for the brain-specific phenotype of SGCE mutation carriers. One explanation could be that brain regions which are not affected are rescued by expression of the maternally imprinted allele. Therefore, we analyzed the SGCE imprinting throughout the human brain with a MS-HRM assay. We observed that the imprinting pattern was maintained in all brain regions and tissues tested (Supplementary Fig. 2). Results of the MS-HRM assay were confirmed by cloning and direct sequencing of a subset of samples. Only minor variations in the number of methylated/unmethylated CpGs were detected by cloning but not by the MS-HRM assay.

SGCE mRNA expression by ultra deep amplicon sequencing

Different SGCE isoforms have been reported so far, but little is known about their distribution and expression levels throughout the brain. To qualitatively and quantitatively characterize alternatively spliced SGCE exons, we performed a systematic analysis of SGCE cDNA by ultra deep amplicon sequencing. This technique provides a powerful and accurate tool to investigate relative mRNA expression levels and detects rare and unknown splice events. To cover all exon-exon boundaries, four overlapping SGCE amplicons were made (exon 1 to 5, exon 4 to 6, exon 5 to 9, exon 7 to 12) and sequenced with a 454 GS FLX sequencer. Ultra deep sequencing was achieved by aiming for 10 000 sequence reads per amplicon (average (± SD): 11 107 (±4 273) reads, Supplementary Table 1).

SGCE mRNA isoforms

To get an overview of all alternative splicing events and their tissue specificity, we analyzed alternative splicing events of the entire SGCE gene in one control subject (Subject 1) in the primary somatosensory cortex (SM1), heart and blood. Twenty-three different splicing events of the SGCE gene were detected, but only four of them occurred at frequencies above 1%: exon 1c and the known alternatively spliced exons 2, 8 and 11b (Fig. 1B, Table 2). In-frame exon 1c was expressed in brain (5.7%), but also in non-brain tissue (2% -2.3%). The in-frame exon 2 showed an overall high inclusion level in all tissues tested, whereas exon 8 was highly represented in muscle and blood and low in brain. Exon 11b was mainly expressed in brain (34%); it was not expressed in blood and at very low levels in muscle (0.05%). These four major variants result in a partially altered protein coding region and are extended into the last exon. Therefore, we expect these to be translated and stable. Of the low frequency variants only one, exon 4a, retains a long open reading frame. All other mRNA isoforms, except all 11b variants, are expected to result in nonsense mediated decay. [START_REF] Maquat | Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics[END_REF] Splice site predictions and positions of known and novel exons are listed in Table 2. Most of the new low frequent variants lead to a frameshift (17/19) resulting in a premature stop codon and were conserved among primate lineage only (Table 2, Supplementary Table 2). All known constitutive SGCE exons were present to 100% except for exon 11: Skipping of this exon was observed in all tissues and species tested, leading to a premature stop after amino acid 409, albeit at low frequency (brain: 0.8%, muscle: 1.4%, blood: 0.3%). The recently identified brain-specific exon 11c was expressed at very low levels in brain but also non-brain tissue (≤0.7%, Table 2). Also, in mouse and rat brain it was expressed at very low levels (< 2.1%), it was not expressed in zebrafish.

In order, to exclude that these low frequency variants were experimental artifacts, we sequenced a synthetic SGCE RNA using the same procedure. Analysis of 12 000 reads gave only sequence reads identical to the input RNA (data not shown).

Brain region-specific SGCE expression

SGCE exon 11b was the most abundant and a highly conserved brain-specific splice variant. Therefore, we analyzed exon 11b expression in more detail and tested nine different human brain regions of two control subjects and three different organisms (mouse and rat brain, zebrafish) by ultra deep amplicon sequencing.

Exon 11b showed differential expression among the different brain regions in both control subjects. Levels were highest in SM1 and motor cortex (M1), low in the globus pallidus, intermediate in caudate nucleus and substantia nigra, and interindividual variation was found in the cerebellum and putamen (Table 3). In mouse and rat brain, we observed a higher rate of exon 11b inclusion compared to human brain (SM1: 50% in mouse, 73% in rat vs. 35% in human). In whole zebrafish RNA, exon 11b was present in 20% of transcripts.

SGCE mRNA expression by qPCR

Ultra deep amplicon sequencing revealed that exon 11b is the major brain-specific alternatively spliced SGCE exon and that it is differentially expressed in the human brain.

Similar trends in expression levels of exon 11b among the different brain regions were identified, but we also observed interindividual variability for some regions. To further characterize the expression pattern of SGCE transcripts we tested the same brain regions of more control subjects by qPCR (Fig. 2). We found similar trends of exon 11b expression in five control subjects: Expression in the cerebellum was significantly higher compared to globus pallidus (Fig. 2,p<0.001, Friedman test, Dunn's multiple comparison test) and substantia nigra (p<0.05). Also SM1 and pallidum were significantly different (p<0.05).

The total SGCE expression level (all isoforms together) was determined in all five control subjects for the various brain regions. Their means did not differ significantly in the different brain regions (p=0.5), suggesting that high exon 11b expression is indeed due to differential isoform expression and not only due to differences in total SGCE expression levels. Overall, SGCE and SGCE exon 11b expression levels varied among individuals.

Normalization with reference genes GAPDH or eEF-1 showed comparable results. Furthermore, the 454 sequencing and qPCR results are in support of each other (Supplementary Fig. 1).

Localization of SGCE mRNA isoforms in human brain

In order, to study SGCE mRNA localization of the major brain-specific and the ubiquitously expressed transcripts we performed isoform-specific ISH in human brain (Fig. 3). SGCE exon 11b and total SGCE expression was most prominent in neurons in all regions tested: high signal in the cerebral cortex, cerebellum (Purkinje cells, dentate nucleus), hippocampus (pyramidal neurons of all CA regions) and moderate signal in the mesencephalon (substantia nigra) and basal ganglia. There was only a faint signal in glial cells and no staining in the white matter. The exon 11b probe did not detect a signal in skeletal muscle, heart, kidney and liver, in contrast to the SGCE probe targeting all isoforms. Probe specificity was confirmed with brain tissue of a SGCE null mutation carrier (c.835_839del, p.Lys280SerfsX16) showing no staining for both probes, whereas it stained for control genes (CD68, TSEN54).

In view of the localized expression of SGCE in the cerebellum, we tested whether the interindividual variation of SGCE exon 11b expression in the cerebellum is due to variation in Purkinje cell number in the tissue examined. qPCR analysis of Purkinje cell marker Calbindin-D28k showed no correlation with SGCE expression (data not shown).

Mutation analysis of novel SGCE exons

We performed mutation analysis in our cohort of definite M-D patients where no mutation was identified [START_REF] Ritz | Myoclonus-dystonia: clinical and genetic evaluation of a large cohort[END_REF] and sequenced novel alternatively spliced SGCE exons. This included all new in-frame exons (1c, 4a) and frameshift exon 11b with all extended versions (exon 11d, 11c, 11f). Also, frameshift exons 3b and 3d were sequenced since their combination was observed in a transcript, and maintains the reading frame. No mutations were identified in the 12 patients analyzed.

Discussion

Mutations in SGCE lead to M-D, but how mutations in this ubiquitously expressed gene result only in neurological impairments is unknown. In this study, we propose a link between the cerebellum and the M-D phenotype. M-D is an autosomal-dominant disease with a reduced penetrance due to maternal imprinting. Monoallelic paternal SGCE expression has been shown in human blood leukocytes and in human and mice brain. 15;18 Several genes are known to show tissuespecific imprinting [START_REF] Albrecht | Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons[END_REF] and brain cell-type or region-specific imprinting. [START_REF] Yamasaki | Neurons but not glial cells show reciprocal imprinting of sense and antisense transcripts of Ube3a[END_REF] The latter has not yet been investigated for SGCE, but is important to address, since only regions in which the imprint is maintained can be involved in the M-D pathogenesis. In this study, we show that the SGCE imprinting pattern is retained in different human brain regions and different human tissues (muscle, blood) similar to what has been shown in mice. [START_REF] Yokoi | Exclusive paternal expression and novel alternatively spliced variants of epsilon-sarcoglycan mRNA in mouse brain[END_REF] Thus, the brainspecific M-D phenotype cannot be explained by a brain region-specific or tissue-specific imprinting. Since not all M-D patients have mutations in SGCE, silencing of SGCE expression due to imprinting defects could also be a possible disease mechanism. We tested our cohort of patients in which no mutation was identified and found a normal imprint in blood. Imprinting defects in the brain cannot be excluded, however.

More likely explanations for the brain-specific symptoms in M-D are the presence of a brain-specific SGCE isoform with a brain-specific function or a brain-specific function for ubiquitous SGCE. Our analysis of SGCE expression in human brain using ultra deep amplicon sequencing shows the presence of 23 alternatively spliced exons, of which 19 at very low frequencies (Table 2). Five of the 23 events retained an open reading frame and are expected to be translated (exon 1c, 2, 4a, 8, and 10). Exon 1c and 4a have not been identified thus far. The majority of the new splice variants have splice sites implying involvement of the splicing machinery. However, most of the new isoforms lead to a frameshift and a premature stop codon which makes them likely a target for nonsense mediated decay. Despite the overall low frequency of all new exons, we cannot exclude that they contribute to the physiological function of SGCE.

Of the four major alternatively spliced exons (exon 1c, 2, 8, 11b), exon 11b was of most interest with respect to the disease: It has a brain-specific expression pattern, was highly conserved and expressed at high levels. Exon 8 expression was low in brain (7.2%) compared to heart and muscle (96%), but its expression in brain appears to be variable. We further analyzed expression of SGCE exon 11b throughout the human brain. We identified differential expression of exon 11b in human brain areas, whereas total SGCE expression levels did not differ. Quantitative PCR analysis confirmed this observation and revealed trends: A consistently high expression in the cerebellum, moderate to high expression in SM1, moderate expression in substantia nigra and putamen and consistently low levels in globus pallidus. We observed a high interindividual variability of exon 11b expression in the cerebellum. Interindividual differences in expression of isoforms could account for this observation 24;25 .

We show that exon 11b is the major brain-specific alternatively spliced SGCE exon and that it is differentially expressed in five control subjects. Its expression pattern is compatible with a role for the brain-specific SGCE isoform in the cerebellum and a link to M-D. This hypothesis is supported by recent literature: (1) there is a shift from dysfunction of the basal ganglia towards involvement of the cerebellum in dystonia pathogenesis; [START_REF] Jinnah | A new twist on the anatomy of dystonia: the basal ganglia and the cerebellum?[END_REF] (2) several animal models linked dystonia to abnormal cerebellar signaling and cerebellar defects, and cerebellar lesions leading to dystonia have been observed in patients; 26;27 (3) fMRI analysis of genetically confirmed M-D patients suggests involvement of thalamus and cerebellum (dentate nucleus) [START_REF] Nitschke | Functional MRI reveals activation of a subcortical network in a 5-year-old girl with genetically confirmed myoclonusdystonia[END_REF] and involvement of different cortical areas and cerebellum. [START_REF] Beukers | Disorganized sensorimotor integration in mutation-positive myoclonus-dystonia: a functional magnetic resonance imaging study[END_REF] A role of the cerebellum in M-D is in contrast with striatal changes on dopamine levels in SGCE deficient mice and a reduced striatal D2 receptor binding in M-D patients. 30;31 Moderate expression levels of the major brain-specific SGCE isoform in the striatum and lowest expression levels in the globus pallidus were found in all control subjects. We propose that observed striatal changes may be secondary due to abnormal cerebellar signaling. This is supported by an anatomical link between the cerebellum and the basal ganglia, namely projections from the dentate nucleus to the striatum, [START_REF] Hoshi | The cerebellum communicates with the basal ganglia[END_REF] and by cerebellar lesions or stimulations that have been shown to alter striatal dopamine signaling. [START_REF] Nieoullon | Release of dopamine in both caudate nuclei and both substantia nigrae in response to unilateral stimulation of cerebellar nuclei in the cat[END_REF][START_REF] Tranchant | Cerebellar cavernous angioma, cervical dystonia and crossed cortical diaschisis[END_REF][START_REF] Neychev | The basal ganglia and cerebellum interact in the expression of dystonic movement[END_REF] Our isoform-specific ISH showed that the major brain-specific SGCE transcript is highly expressed in Purkinje cells and neurons of the dentate nucleus in the cerebellum. No signals were detected in the cerebellar granule cell layer in the cerebellum as it has been shown in mouse brain. [START_REF] Nishiyama | Identification and characterization of epsilon-sarcoglycans in the central nervous system[END_REF] Interestingly, alcohol consumption alleviates the symptoms in M-D and the cerebellum reacts very sensitively to alcohol. [START_REF] Volkow | Effects of acute alcohol intoxication on cerebral blood flow measured with PET[END_REF] Also, symptoms of patients with essential tremor, a common neurological movement disorder with suggested involvement of the cerebellum, [START_REF] Dupuis | Homolateral disappearance of essential tremor after cerebellar stroke[END_REF][START_REF] Louis | Essential tremor associated with pathologic changes in the cerebellum[END_REF][START_REF] Louis | Essential tremor: evolving clinicopathological concepts in an era of intensive post-mortem enquiry[END_REF] are known to respond to alcohol as seen in M-D.

We propose that the loss of function of the brain-specific SGCE isoform underlies the exclusively neurological M-D phenotype. We hypothesize that the general function of the abundant SGCE isoform is redundant or not essential since no non-neurological symptoms have been reported. The brain-specific protein may have a unique function and cannot be replaced.

Titles and legends to figures

Figure 1 :

 1 Overview of novel alternatively spliced exons identified by 454 ultra deep sequencing. A) SGCE gene including all known exons. Constitutive exons are depicted in black, alternatively spliced exons in grey. B) SGCE gene including novel alternatively spliced exons (in white). Asterisks represent novel in-frame exons. In-frame exons were observed with a higher frequency (range: 0.3-5.7%) than frameshift exons (range: 0.01-3%). Relative position of the exons and sizes of exons are correct, but exon sizes are not represented in the same scale as the introns.

Figure 2 :

 2 Figure 2: SGCE expression levels (qPCR). Displayed are expression levels for total

Figure 3 :

 3 Figure 3: Localization of total SGCE transcripts and those containing exon 11b.

Tables 1- 3

 3

  brain (SM1), heart and blood of control subject 1 in percent, splice sites according to www.fruitfly.org, NNSPLICE 0.9 version (minimum score for 3'/ 5'splice site 0.5): splice donor (D)/acceptor (A), frameshift (FS) or in-frame (IF) exons, isoform subject to nonsense mediated decay (NMD), exon size in bp, nucleotide position according to NG_008893.1. Constitutive exons (100% expression) are not shown. *: Exons that were sequenced in definite M-D patients where no mutation could be identified.

Table 1 : Sample material

 1 Cerebellum, GP: Globus Pallidus, Hi: Hippocampus, M1: Primary Motor Cortex, NC: Caudate Nucleus, Pu: Putamen, SM1: Primary Somatosensory Cortex, SN: Substantia Nigra, Th: Thalamus, He: Heart, MP: M.Psoas, na: not available

	Subject	Gender	Age (years)	Post-mortem (h)	Brain tissue	Other	Assay
	1	male	77	7	Cb GP Hi M1 NC Pu SM1 SN Th	He MP	454 qPCR
	2	male	76	8	Cb GP Hi M1 NC Pu SM1 SN Th	na	454 qPCR
	3	female	76	5	na	He MP	454
	4	male	74	5	Cb GP Hi M1 NC Pu SM1 SN Th	na	qPCR
	5	female	88	12	Cb GP Hi M1 NC Pu SM1 SN Th	na	qPCR
	6	female	45	24	Cb GP Hi M1 NC Pu SM1 SN Th	na	qPCR
	Abbreviations: Cb:

Table 2 : Known and new alternatively spliced SGCE exons revealed by screening the entire SGCE gene in one control subject with ultra deep sequencing.

 2 Additional information of alternatively spliced SGCE exons include: occurrence of respective exons in

Acknowledgements

We thank Edward J Bradley for assistance with 454 sequencing and Dr Kees Fluiter for help with ISHs. We thank Dr. Anneloor ten Asbroek for helpful discussion and critical reading of the manuscript. Tissue from the M-D mutation carrier was obtained from the Brain and Tissue Bank for Developmental Disorders at the University of Maryland under contracts N01-HD-4-3368 and N01-HD-4-3383.

Fundings

This study was supported by Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) VIDI (project 0160.056.333) and by the Prinses Beatrix Fund.

Supplementary Fig. 2

Supplementary Tables 12Supplementary Localization of brain-specific SGCE in small and large striatal neurons.

Higher magnifications of ISHs for detection of SGCE exon 11b transcripts as an example to illustrate neuronal staining in the putamen. Scale bar represents 50µm.

Optional figure 1:


Georg M Singewald

Alesja Rjabokon

Nicolas Singewald

Dr Karl Ebner
email: karl.ebner@uibk.ac.at

The modulatory role of the lateral septum on neuroendocrine and behavioral stress responses

Keywords: forced swimming, HPA axis, ACTH, corticosterone, glucocorticoids, serotonin

The lateral septum (LS) has been shown to play a key role in emotional processes and stress responses. However, the exact role of the LS upon stress modulation is not clear, since previous lesion studies mostly used electrolytic lesions, thereby destroying the whole septal area including medial components and/or fibres of passage. The aim of the present study was therefore, to investigate the effects of selective excitotoxic ablation of the LS on neuroendocrine and behavioral stress responses in rats. Bilateral ibotenic-acid lesions of the LS increased hypothalamopituitary-adrenocortical (HPA) axis responses to forced swim stress indicated by enhanced plasma ACTH and corticosterone responses and higher stress-induced c-Fos-like immunoreactivity in the paraventricular hypothalamic nucleus. Moreover, LSlesioned animals displayed a more passive coping-style in the forced-swim-test indicated by increased floating and reduced struggling/swimming-behavior compared to sham-lesioned controls. Interestingly, intraseptal corticosteroid-receptor blockade modulated behavioral stress-coping but failed to change HPA-axis stress responses.

Further experiments aimed at elucidating underlying neurochemical mechanisms revealed that intraseptal administration of the selective 5-HT 1A receptor antagonist WAY-100635 increased and prolonged stress-induced ACTH and corticosterone levels mimicking lesion effects, while the agonist 8-OH-DPAT suppressed HPA-axis activity facilitating the inhibitory role of the LS. Additionally, 8-OH-DPAT injected animals showed increased active and decreased passive coping strategies during forced swimming suggesting antidepressant efficacy. Taken together, our data suggest that the LS promotes active stress coping behavior and is involved in a HPAinhibitory mechanism that is at least in part mediated by septal 5-HT 1A receptors and does not involve a glucocorticoid mediated feedback mechanism.

Introduction

Considerable evidence suggests that a significant element in the etiology of numerous psychiatric disorders including depression and posttraumatic stress disorder is a dysregulated stress hormone function [START_REF] Kloet | Stress and the brain: from adaptation to disease[END_REF][START_REF] Krishnan | The molecular neurobiology of depression[END_REF]. The release of stress hormones such as glucocorticoids is regulated mainly by the hypothalamo-pituitary-adrenocortical (HPA) axis and it is well documented that glucocorticoids play an important role in the adaptation of the organism to stress, including the coordination of appropriate behavioral response to stressors [START_REF] Mcewen | Physiology and neurobiology of stress and adaptation: central role of the brain[END_REF]. The importance of maintaining glucocorticoid secretion within tolerable limits requires efficient mechanisms for inhibiting neuroendocrine stress responses. To avoid excessive stress reactions glucocorticoid levels are regulated by a negative feedback mechanism, which mainly acts at the level of hypothalamus and pituitary [START_REF] Keller-Wood | Corticosteroid inhibition of ACTH secretion[END_REF][START_REF] Kloet | Brain corticosteroid receptor balance in health and disease[END_REF].

In parallel with steroid feedback there is also evidence for the existence of further glucocorticoid-independent inhibitory pathways that might emanate from neuronal sources including GABAergic neurons from multiple brain areas including neurons scattered in the immediate surroundings of the hypothalamic paraventricular nucleus (PVN) [START_REF] Herman | Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness[END_REF][START_REF] Herman | Role of GABA and glutamate circuitry in hypothalamo-pituitary-adrenocortical stress integration[END_REF][START_REF] Herman | Limbic system mechanisms of stress regulation: Hypothalamo-pituitary-adrenocortical axis[END_REF][START_REF] Cullinan | Functional role of local GABAergic influences on the HPA axis[END_REF]. Additional studies have now provided evidence that the lateral septum (LS) is involved in HPA axis activity modulation [START_REF] Herman | Neuronal circuit regulation of the hypothalamo-pituitary-adrenocortical stress axis[END_REF][START_REF] Herman | Limbic system mechanisms of stress regulation: Hypothalamo-pituitary-adrenocortical axis[END_REF]. However, the exact role of the LS in HPA axis regulation is presently obscure, because of inconsistent findings from prior studies. For example, basal plasma corticosterone levels following septal lesions are elevated in some studies [START_REF] Usher | Pituitary adrenal function after small and large lesions in the lateral septal area in food-deprived rats[END_REF][START_REF] Dobrakovova | Changes of plasma and adrenal catecholamines and corticosterone in stressed rats with septal lesions[END_REF] but not in others [START_REF] Seggie | Septal lesions and resting adrenal function. A possible explanation of conflicting findings[END_REF][START_REF] Seggie | Adrenal stress responses following septal lesions in the rat[END_REF][START_REF] Uhlir | The effect of septal lesions on the threshold of adrenal stress response[END_REF][START_REF] Dobrakovova | Changes of plasma and adrenal catecholamines and corticosterone in stressed rats with septal lesions[END_REF][START_REF] Seggie | Differential responsivity of corticosterone and prolactin to stress following lesions of the septum or amygdala: implications for psychoneuroendocrinology[END_REF]. Moreover, the impact of LS-neurons on the expression of anxiety-related behaviors are similar controversial as both reduced [START_REF] Pesold | Excitotoxic lesions of the septum produce anxiolytic effects in the elevated plus-maze and the shock-probe burying tests[END_REF][START_REF] Menard | Lateral and medial septal lesions reduce anxiety in the plusmaze and probe-burying tests[END_REF] as well as increased anxiety levels [START_REF] Albert | The septal forebrain and the inhibitory modulation of attack and defense in the rat. A review[END_REF][START_REF] Yadin | The role of the lateral septum in anxiolysis[END_REF] were found after septal lesions. These discrepant findings might be related to the fact that most of the above mentioned lesion studies used large electrolytic lesions, thereby damaging both lateral and medial divisions of the septal area as well as extensive portions of the fornix system and thus fibres passing through this region. So far, only one study investigated the effects of selective LSlesions on the HPA-axis activity by using the excitotoxic lesion agent ibotenic acid, that spares fibres of passage [START_REF] Herman | Neuronal circuit regulation of the hypothalamo-pituitary-adrenocortical stress axis[END_REF]. However, since this study was performed under basal conditions only, where no modulatory function of the LS was found, the specific role of LS neurons under stress conditions is still unknown.

The aim of the present study was to investigate the effects of axon-sparing excitotoxic lesions of LS neurons on neuroendocrine and behavioral stress responses. Moreover, we examined whether the LS is involved in the glucocorticoidmediated negative feedback mechanism, as a high density of glucocorticoid receptors was found in the LS of rodents and primates [START_REF] Morimoto | Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain: an immunohistochemical and in situ hybridization study[END_REF][START_REF] Patel | Glucocorticoid and mineralocorticoid receptor mRNA expression in squirrel monkey brain[END_REF]. Finally, we aimed to study the underlying neurochemical mechanisms of how LS neurons modulate neuroendocrine and behavioral stress responses.

Materials and Methods

Animals

All experiments were carried out on adult male Sprague-Dawley rats (250 -320g).

Before use, the animals were housed in groups of four to six under controlled laboratory conditions (12:12 h light/dark cycle with lights on at 07:00, 21 ± 1°C, 60% humidity, pelleted food and water ad libitum) for at least 1 week after delivery from the supplier. All experiments were approved by the local Ethical Committee on Animal Care and Use of the Austrian governmental body.

Surgery

All surgeries were conducted under sodium pentobarbital (40 mg/kg, i.p.) and ketamine (50 mg/kg, i.p.) anaesthesia. Moreover, before incision the local anaesthetic xylocaine (AstraZeneca, Vienna, Austria) were injected on the head surface of animals. Postoperatively, rats received a one-time injection of buprenorphine (0.03 mg/kg i.m.).

Excitotoxic lesions: Excitotoxic lesions were produced bilaterally by microinjections of ibotenic acid (Sigma-Aldrich, Steinheim, Germany) into the LS with a Hamilton microsyringe stereotaxically inserted according to a brain atlas [START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF]. The implantation coordinates were: 0.6 mm rostral to bregma, 0.8 mm lateral to the midline. To ensure ablation that implies most of the LS, lesions were placed 7.0 and 5.5 mm ventral to skull surface. Each injecting point was infused with 0.5 µl of ibotenic acid (5 µg/µl) during a time period of 5 min. After injections the injection cannula was left in position for an additional 5 min to facilitate drug diffusion and minimize dragging of the injected liquid along the injection tract. Sham-lesioned animals received the same surgical treatment as a burr hole was drilled in the skull and the dura was removed in the location dorsal to LS lesion placements, except that no ibotenic acid was applied.

Implantation of microinjection cannulae:

For microinjection experiments, 25 gauge stainless steel cannulae (15 mm length) used as guide cannulae were bilaterally implanted with their tip 2 mm above the LS (implantation coordinates: 0.6 mm rostral to bregma, 1.6 mm lateral to the midline, 3.7 mm below the surface of the skull with an angle of 10° to avoid sagittal sinus damage) and fixed to the skull with two jeweller's screws and dental cement.

Implantation of a microdialysis probe:

A homemade, U-shaped microdialysis probe was stereotaxically implanted two days prior to experiments as previously described [START_REF] Ebner | Substance P in the medial amygdala: emotional stress-sensitive release and modulation of anxietyrelated behavior in rats[END_REF] with its tip reaching the right LS (implantation coordinates: 0.6 mm rostral to bregma, 1.6 mm lateral to the midline, 7.0 mm below the surface of the skull with an angle of 10° to avoid sagittal sinus damage). The probe consisting of an 18 kDa dialysis membrane (Hemophan, Membrana, Wuppertal, Germany) with an active surface length of 2 mm was fixed to the skull with two jeweller's screws and dental cement. The two endings of the probe were attached to 5-cm-long pieces of polyethylene tubing for connection with the infusion pump.

Implantation of a jugular venous catheter:

A silastic-tipped vinyl catheter was inserted into the left jugular vein, routed under the skin and exteriorized at the neck of the animal as described previously [START_REF] Ebner | Neuroendocrine and behavioral response to social confrontation: residents versus intruders, active versus passive coping styles[END_REF]. The wounds were closed using metal clips. The catheter was filled with sterile saline containing gentamycin (30.000 IU/rat, Centravet, Bad Bentheim, Germany) and flushed with the same solution 2 days after surgery. On the day of the experiment the catheters were connected to 1ml plastic syringes via approximately 40 cm long pieces of PE-50 tubing 2h before starting the experiment. Blood sampling through a pre-implanted jugular vein catheter allows repeated blood sampling from conscious, freely moving rats without restraining animals.

Experimental protocol

After surgery, rats were housed individually in transparent plexiglas cages until testing. Operated animals were allowed to recover from surgery for 2-3 days during they were handled for 3 min twice daily to familiarize them with the experimental procedure and to minimize nonspecific stress responses during the experiments. At least 16 hours before experiments, animals were placed in the experimental room and allowed to habituate. All experiments were performed between 07:00 and 16:00.

Exposure to the stressor was always completed between 11:00 and 14:00 to minimize circadian rhythm-related variations in stress responses.

Effects of LS-lesions on stress responses

The experiment started with the collection of a first blood sample (0.3 ml) under basal conditions, 30 min prior to stressor. After sampling of the basal blood sample, lesioned and sham-lesioned animals were exposed to forced swimming according to the procedure described previously [START_REF] Ebner | Neurokinin 1 receptor antagonism promotes active stress coping via enhanced septal 5-HT transmission[END_REF]. Briefly, the rats were forced to swim for 5 min in a square plastic tank (40 cm in diameter) filled to a depth of 30 cm with tap water (20 ± 1°C). During the forced swimming session the behavior was recorded by a video system and scored by a trained observer blind to the animals' treatment quantifying absolute time measurements. The behavior of the animals was assigned to one of the three following behavioral categories: (1) struggling, defined as movements during which the forelimbs broke the water's surface; (2) swimming, defined as movement of the animal induced by movements of the fore and hind limbs without breaking the water surface; and (3) floating defined as the behavior during which the animal used limb movement just to keep its equilibrium without any movement of the trunk [START_REF] Ebner | Neurokinin 1 receptor antagonism promotes active stress coping via enhanced septal 5-HT transmission[END_REF]. After the 5-min swimming session, animals were gently dried using a towel and returned to their home cage. To determine the time course of plasma ACTH and corticosterone release in response to forced swim stress, additional blood samples were collected 10, 30, and 60 min after onset of the stressor. Sampled blood volumes were immediately replaced with an equal volume of heparinized saline.

Effects of intraseptal administration of selective corticosteroid receptor antagonists on stress responses

In a separate group of animals, the stylets of guide cannulae were replaced by microinjection cannulae (31 gauge), which were 2 mm longer than the guide cannulae, thus reaching the LS. Injection cannulae were connected to a syringe mounted on a microinfusion pump (TSE-Systems, Bad Homburg, Germany) and filled either with artificial cerebrospinal fluid (aCSF; pH 7.2; 140 mM NaCl, 3.0 mM KCl, 1.25 mM CaCl 2 , 1.0 mM MgCl 2 , 1.2 mM Na 2 HPO 4 , 0.3 mM NaH 2 PO 4 , and 3.0 mM glucose) or aCSF containing the corticosteroid receptor antagonists mifepristone or RU-28318 (30 and 150 µM; Tocris Bioscience, Bristol, UK). The dose range of RU-28318 and mifepristone used in our experiments was based on previous studies demonstrating a modulatory effect on HPA axis activity as well as emotional behavior in rats after intracerebroventricular, intrahippocampal or intraseptal injections [START_REF] Ratka | On the role of brain mineralocorticoid (type I) and glucocorticoid (type II) receptors in neuroendocrine regulation[END_REF][START_REF] Korte | Anxiolytic-like effects of selective mineralocorticoid and glucocorticoid antagonists on fear-enhanced behavior in the elevated plus-maze[END_REF][START_REF] Van Haarst | Facilitation of feedback inhibition through blockade of glucocorticoid receptors in the hippocampus[END_REF][START_REF] Calvo | Glucocorticoid and mineralocorticoid receptors are involved in the facilitation of anxiety-like response induced by restraint[END_REF][START_REF] Gesing | Psychological stress increases hippocampal mineralocorticoid receptor levels: involvement of corticotropin-releasing hormone[END_REF][START_REF] Calfa | Glucocorticoid receptors in lateral septum are involved in the modulation of the emotional sequelae induced by social defeat[END_REF]. After a habituation period of 60 min, experiment started with the collection of two blood samples (0.3 ml) under basal conditions, 30 and 15 min before stress exposure. Bilateral infusions of the corticosteroid receptor antagonists into the LS started 12 min before onset of the stressor. The injection time of 7.5 min with a defined flow rate of 0.2 µl/min achieved a total volume of 1.5 µl (=20 and 100 ng) for each injection side followed by 2.5 min where the injectors were kept in place to allow absorption of the injection bolus into the tissue. After injections another blood sample was taken and one minute later animals were exposed to the forced swimming procedure for 5 min as described above. During the swim session behavioral output was scored and analyzed as described above. Additional blood samples were collected 10, 30, and 60 min after onset of the stressor and again equal volume of heparinized saline was reinfused.

Effects of intraseptal administration of 5-HT 1A receptor ligands on stress responses

In a further series of experiments, the selective 5-HT 1A receptor agonist 8-OH-DPAT (10 µg/side; Sigma-Aldrich, Steinheim, Germany) was bilaterally microinjected into the LS as described above. In order to intraseptally administer the selective 5-HT 1A receptor antagonist WAY-100635 (Sigma-Aldrich) over an extended period of time with no further perturbation to the surrounding tissue [START_REF] Bourne | Intracerebral microdialysis: 30 years as a tool for the neuroscientist[END_REF] we decided to infuse the compound via retrodialysis into the LS as described previously [START_REF] Ebner | Neurokinin 1 receptor antagonism promotes active stress coping via enhanced septal 5-HT transmission[END_REF]. Briefly, at the day of experiment the microdialysis probe was connected to a syringe mounted onto a superfusion pump and dialysed with aCSF. After an equilibration period of 120 min, experiment started with the collection of two blood samples (0.3 ml) under basal conditions, 120 and 60 min before stress exposure, followed by infusion of WAY-100635 (10 µM) in aCSF or aCSF alone (controls) with a flow-rate of 3.3 µl/min. Thirty minutes after the start of retrodialysis another blood sample was collected. The drug reached the LS after approximately 5-6 min and forced swimming was applied 30 min later as described above. During the swim session behavioral output was scored and analyzed as described above and afterwards three additional blood samples were collected 10, 30 and 60 min (sample 4-6) after onset of the stressor.

ACTH and corticosterone radioimmunoassay

Plasma ACTH and corticosterone concentrations were measured by radioimmunoassay using commercially available kits (MP Biomedicals, Orangeburg, NY, USA) with an intra and inter-assay variability of less than 10% and a lower limit of detection of 6 pg/ml for ACTH and 8 ng/ml for corticosterone.

Immunocytochemistry

Two hours after stress exposure, lesioned and sham-lesioned animals were deeply anaesthetized with an overdose of sodium pentobarbital (200 mg/kg) and transcardially perfused with 100 ml of 0.9% saline followed by 100 ml of 4% paraformaldehyde in 0.1 mol/l phosphate buffered solution (PBS, pH 7.4). The 2 h time point represents the interval at which maximal c-Fos protein expression has been reported in acute stress paradigms [START_REF] Viau | Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stress: rapid publication[END_REF]. Brains were then removed, postfixed for 2 h, and cryoprotected at 4°C overnight in 10% sucrose (in 0.1 M phosphate buffered saline, pH 7.4). Coronal sections (40 μm) were cut through the PVN using a Cryostat (Leica CM 1850, Leica-Microsystems, Nussloch, Germany) and collected in PBS. The sections were processed for c-Fos-like immunoreactivity as described previously [START_REF] Smith | Neurochemical identification of fos-positive neurons using two-colour immunoperoxidase staining[END_REF][START_REF] Buller | Differential recruitment of hypothalamic neuroendocrine and ventrolateral medulla catecholamine cells by nonhypotensive and hypotensive hemorrhages[END_REF].

Briefly, brain sections were incubated for 48 h in rabbit anti-Fos (1:50.000, Santa Cruz, CA, USA), followed by a 2h incubation in a biotinylated donkey anti-rabbit (1:300, Jackson ImmunoResearch, West Grove, PA, USA). Sections were then incubated for a further 2 h in a solution of avidin-biotin-horseradish peroxidase complex (ABC Vector Elite Kit, Burlingame, CA, USA) before being exposed to a nickel 3,3-diaminobenzidine solution to allow visualization of the horseradish peroxidase activity. Cells containing a nuclear brown-black reaction product were considered as c-Fos positive cells. The reaction was terminated once an optimal contrast between specific cellular and nonspecific background labelling was reached.

To minimize variations in immunolabelling, sections from each experimental group were processed simultaneously. Sections were then mounted on chrome-alum subbed slides, dehydrated in alcohol, cleared in xylene and cover-slipped.

Histology

At the end of the experiments animals were killed by an overdose of pentobarbital and their brains were removed. For histological verification of either the extension of LS lesions -on the basis of gliosis and neuronal cell loss (Fig. 1) -or placement of microinjection cannulae and microdialysis probes (Fig. 2), brains were sectioned using a Cryostat and 20-40 µm coronal sections were stained with cresyl violet.

Lesion verification and judgment of successful implantation of cannulae and probes into the LS was made before analyzing neuroendocrine and behavioral experiments.

Statistics

Experimental subjects were included in the statistical analysis only if the lesions, microinjection or microdialysis cannulae were confirmed to be localized within the LS.

Statistical analysis was performed using a computer software package (GB-Stat 6.0, Dynamic Microsystems, Silver Springs, USA). Plasma concentrations of ACTH and 13 corticosterone were analyzed using a two-way ANOVA (treatment/lesion x time) with repeated measures on the last factor followed by appropriate post-hoc analysis.

Behavioral data and counts of c-Fos-positive nuclei within the PVN after stress exposure were analyzed using Student's two-tailed t-test comparing lesioned or drug treated animals with respective controls. Anatomical boundaries defining the subareas of interest within the PVN were evaluated with reference to morphological and cytoarchitectonic organization of the PVN [START_REF] Swanson | Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms[END_REF].

Data are presented as means ± SEM. Statistical significance was accepted if p<0.05.

Results

Lesion placements

Lesions were evaluated with reference to morphological and cytoarchitectonic organization of the LS [START_REF] Alonso | Organization of the septal region in the rat brain: a Golgi/EM study of lateral septal neurons[END_REF][START_REF] Risold | The septal region[END_REF]. Three subregions were of primary interest: the dorsal, the intermediate and the ventral part (Fig. 1A, left panel). Lesion placement and extent was verified by light microscopic examination of cresyl violet-stained sections showing substantial cell degeneration and cell loss resulting from ibotenic acid injections in lesioned compared to sham-lesioned animals (Fig. 1A, right panel). Analysis of LS-lesions indicated significant damage in all subregions as well as over the entire rostro-caudal extension of the LS (Fig. 1B).

Lesion effects on neuroendocrine stress response

Exposure to forced swimming caused an increase in plasma ACTH and corticosterone levels in both LS-lesioned animals and sham-lesioned controls (Fig. 3A). Statistical analysis of ACTH levels by two-way ANOVA revealed a significant effect of the main factors (lesion: F 1,22 =8.56, p=0.0078; time: F 3,66 =82.87, p<0.0001) as well as a significant interaction between main factors (F 3,66 =3.24, p=0.0275). Post hoc analysis indicated significant differences of ACTH levels between lesioned animals and controls immediately after stress exposure with a higher ACTH response to forced swimming at 10 (p<0.01) and 30 min (p<0.05) after onset of the stressor in lesioned animals compared to sham-lesioned controls. In contrast, basal levels of ACTH did not vary as a function of lesion status (Fig. 3A, -30 min).

Plasma corticosterone levels showed similar main effects (lesion: F 1,22 =4.97, p=0.03; time: F 3,66 =69.89, p<0.0001) and interaction of main factors (F 3,66 =2.94, p=0.03).

Lesion status differentially affected the magnitude and longevity of the response, with lesioned animals displaying higher and prolonged plasma corticosterone levels after forced swim stress than sham-lesioned controls. Basal levels of corticosterone were not different between groups.

Lesion effects on stress-induced PVN activation

Next we examined whether ablation of the LS is associated with an increased neuronal activity in the PVN, the apex of HPA axis regulation. Therefore, we compared the stress-induced expression of the immediate early gene product c-Fos in the PVN of LS-lesioned animals and controls. As shown in Fig. 3B, sham-lesioned animals subjected to forced swim stress displayed moderate numbers of c-Fospositive cells in the PVN. Moreover, stress-induced c-Fos expression in the PVN especially in the medial parvocellular part of the PVN (mpPVN) was considerably enhanced (by 140%) in LS-lesioned animals compared to sham-lesioned controls (p<0.001).

Lesion effects on behavioral stress response

To determine the role of LS neurons on stress-related behavioral regulation we monitored stress coping behavior during forced swimming in lesioned animals and controls. In this experiment we found that LS-lesions significantly affected the animals' behavior during forced swimming. As illustrated in Fig. 4, LS-lesioned animals compared to sham-lesioned controls showed increased floating behavior (p<0.001) and a decreased time spent with active coping such as struggling and swimming (p<0.001).

Effects of intraseptal administration of selective corticosteroid receptor antagonists on stress responses

In a further series of experiments, we aimed to clarify whether HPA inhibitory effects of the LS are mediated by septal glucocorticoid and/or mineralocorticoid receptors.

Bilateral infusion of the selective glucocorticoid receptor antagonist mifepristone into the LS at different concentrations (20 and 100 ng) had no effect on HPA axis activity since neither basal nor stress-induced ACTH (Fig. 5A) or corticosterone levels (Fig. 5B) differed between mifepristone and aCSF injected control animals. Moreover, intraseptal administration of the selective mineralocorticoid receptor antagonist RU-28318 had no effect on HPA axis activity. There was no difference either in basal ACTH (basal controls: 14.6 ± 1.6 pg/ml) or corticosterone levels (basal controls: 60.3 ± 15.2 ng/ml) between vehicle injected controls and RU-28318 treated rats. Also, the stress-induced ACTH and corticosterone levels did not differ between controls (peak levels ACTH: 337.4 ± 86.3 pg/ml; CORT: 759.5 ± 64.2 ng/ml) and antagonist treated animals (20 ng RU-28318: ACTH: 320.3 ± 68.1 pg/ml; CORT: 726.1 ± 23.1 ng/ml; 100 ng RU-28318: ACTH: 304.9 ± 60.8 pg/ml; CORT: 767.7 ± 31.7 ng/ml). However, septal glucocorticoid receptor blockade affected the animals' stress coping behavior during forced swimming. As illustrated in Fig. 5C, mifepristone injected animals showed increased struggling and swimming behavior (p<0.01) and decreased time spent floating (p<0.01) compared to aCSF injected controls. In contrast, mineralocorticoid receptor antagonism did not influence stress coping behavior as floating (20ng: 145.8 ± 10.1; 100 ng: 144.5 ± 17.4) or struggling/swimming behavior of RU-28318 treated rats (20ng: 153.2 ± 10.2; 100 ng: 154.1 ± 18.0) did not differ to that of vehicle treated controls (floating: 137.6 ± 8.6; struggling/swimming: 161.8 ± 8.5).

Effects of intraseptal administration of 5-HT 1A receptor ligands on stress responses

As shown in Fig 6, our data indicate significant differences in the plasma ACTH and corticosterone response to forced swimming between 5-HT 1A receptor agonist treated animals and controls. Statistical analysis of ACTH levels by two-way ANOVA showed a significant effect of the factor time (F 5,85 =89.57, p<0.0001) and a significant interaction between main factors (F 5,85 =3.80, p=0.0037). Post hoc analysis revealed that the administration of the selective 5-HT 1A receptor agonist 8-OH-DPAT locally into the LS resulted in a significant attenuation of swim stress-induced ACTH levels.

The attenuation of the plasma ACTH response to forced swimming after intraseptal 8-OH-DPAT administration was evident only at the peak level at 10 min after stressor onset, while at 30 min where ACTH levels were still significantly elevated this difference was not observed. As shown in Fig. 6B, all experimental groups exhibited increased plasma corticosterone levels in response to the 5 min forced swim stress exposure with peak levels at 30 min after stressor onset. Similarly to ACTH, 8-OH-DPAT significantly reduced the stress-induced rise of plasma corticosterone levels.

Statistical analysis by two-way ANOVA revealed significant effects of the main factors (treatment: F 1,17 =6.88, p=0.0178; time: (F 5,85 =71.42, p<0.0001) and a significant interaction between main factors (F 5,85 =3.05, p=0.0137). Notably, attenuated stress-induced corticosterone levels after intraseptal 8-OH-DPAT administration were observed only in the post-stress period with peak corticosterone levels at 30 and 60 min after stressor onset. In contrast, basal ACTH and corticosterone levels were not different between groups. Furthermore, intraseptal administration of 8-OH-DPAT significantly affected the animals´ behavior during forced simming, indicated by increased active coping such as struggling and swimming behavior (p<0.01) and decreased time spent floating (p<0.01) in agonist treated animals compared to controls (Fig. 6C). Fig. 7 illustrates the effects of intraseptal 5-HT 1A receptor blockade on basal and stress-induced ACTH and corticosterone levels. Infusion of the selective 5-HT 1A receptor antagonist WAY-100635 into the LS changed ACTH responses to forced swimming. Statistical analysis by two-way ANOVA showed a significant effect of the main factors (treatment: F 1,10 =11.46, p=0.00; time: F 5,50 =75.02, p<0.0001) as well as a significant interaction between main factors (F 5,50 =7.38, p<0.0001). Post hoc analysis revealed that intraseptal administration of WAY-100635 resulted in higher and prolonged swim stress-induced ACTH levels. Notably, this effect was longlasting and observable even at 60 min after stress exposure because at this time point antagonist treated animals but not controls showed elevated ACTH levels compared to basal. Similarly to ACTH, intraseptal WAY-100635 administration resulted in higher stress-induced corticosterone levels (notably at 30 and 60 min after stressor onset) than in controls with significant main factors (treatment: F 1,10 =38.61, p<0.0001; time: F 5,50 =276.86, p<0.0001) as well as a significant interaction between main factors (F 5,50 =4.41, p=0.002). In contrast, basal ACTH and corticosterone levels were not different between groups. Moreover, intraseptally administered WAY-100635 did not affect the animals´ stress coping behavior in the forced swim test as floating (113.8 ± 15.9) or struggling/swimming behavior (153.2 ± 10.2) of WAY-100635 treated rats did not differ to that of vehicle treated controls (floating: 95.4 ± 9.8; struggling/swimming: 161.8 ± 8.5).

Discussion

The results of the present study demonstrate an important role of the LS in the modulation of behavioral and neuroendocrine stress responses. Ablation of the LS was associated with disinhibited HPA axis stress responses indicated by enhanced plasma ACTH and corticosterone levels and an exaggerated c-Fos expression in the mpPVN. In contrast, lesions did not affect basal plasma hormone levels. The enhanced neuroendocrine stress response in LS-lesioned animals was accompanied by alterations in the stress-coping behavior during forced swimming, indicated by increased floating and reduced struggling/swimming behavior. Additionally, we found that this HPA inhibitory mechanism of the LS is not mediated via septal corticosteroid receptors as intraseptal glucocorticoid and mineralocorticoid receptor blockade failed to change HPA axis activity. However, we identified septal 5-HT 1A receptors to play an important role in this mechanism as intraseptal 5-HT 1A receptor activation suppressed while blockade of these receptors increased and prolonged stressinduced ACTH responses. Moreover, septal 5-HT 1A receptors also seem to be implicated in the modulation of behavioral stress coping as intraseptal administration of a 5-HT 1A receptor agonist increased active and decreased passive stress-coping strategies.

Role of LS neurons on neuroendocrine stress response

In the present work we investigated the effects of selective lesions of the LS on plasma ACTH and corticosterone levels under basal and stress conditions. Notably, we could not observe any differences in basal ACTH or corticosterone levels between LS-lesioned animals and controls. Thus, our data are in contrast to previous findings showing higher corticosterone levels after ablation of the septum suggesting a tonic inhibitory effect of septal neurons on HPA axis activity [START_REF] Uhlir | The effect of septal lesions on the threshold of adrenal stress response[END_REF][START_REF] Bouille | Influence of septal nuclei on basal pituitary-adrenocortical function in birds[END_REF][START_REF] Dobrakovova | Changes of plasma and adrenal catecholamines and corticosterone in stressed rats with septal lesions[END_REF]. Methodological differences might be a possible explanation of this discrepancy. Since these early lesion studies used large electrolytic lesions, thereby destroying the whole septum and extensive portion of the fornix system (see introduction) such kind of destruction can likely lead to a damage of putative inhibitory input to PVN neurons descending through the septal region. Thus, the results of these early studies are difficult to interpret with regard to the specific role of distinct subregions of the septum in HPA axis regulation. In line with our results are data from J.P. Hermans group showing no changes in basal corticosterone levels in rats with selective lesioning of the LS by using the neurotoxic agent ibotenic acid [START_REF] Herman | Neuronal circuit regulation of the hypothalamo-pituitary-adrenocortical stress axis[END_REF].

However, a novel finding in the present study was that ACTH and corticosterone responses to swim stress increased after selective lesioning of LS neurons. In support of this, we observed increased neuronal activation (indicated by an enhancement of c-Fos-positive cells) in the mpPVN, the key region of HPA axis regulation that contains the main neuropeptidergic adrenocorticotropin secretagogues such as CRF and arginine vasopressin [START_REF] Antoni | Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor[END_REF][START_REF] Whitnall | Regulation of the hypothalamic corticotropin-releasing hormone neurosecretory system[END_REF]. Thus, our data suggest an inhibitory role of LS neurons on stress-induced HPA axis activity and extend previous findings demonstrating higher corticosterone levels after electrolytic lesioning of the whole septum in rats [START_REF] Brown | Effect of septal lesions on plasma levels of MSH, corticosterone, GH and prolactin before and after exposure to novel environment: role of MSH in the septal syndrome[END_REF][START_REF] Usher | Pituitary adrenal function after small and large lesions in the lateral septal area in food-deprived rats[END_REF][START_REF] Dobrakovova | Changes of plasma and adrenal catecholamines and corticosterone in stressed rats with septal lesions[END_REF][START_REF] Seggie | Differential responsivity of corticosterone and prolactin to stress following lesions of the septum or amygdala: implications for psychoneuroendocrinology[END_REF]. In contrast to previous studies where only corticosterone levels were measured our study provides information on HPA axis activity at different levels from hypothalamus, pituitary to adrenal level.

Moreover, blood sampling through a jugular catheter as was done in our study offers the advantage of collecting sequential samples to monitor temporal changes in plasma stress hormones during and after stress exposure. Thus, in contrast to previous studies where blood samples were collected by decapitation our study provides some intra-individual time course information.

Next, we examined whether this HPA inhibitory mechanism of the LS is mediated by corticosteroid receptors which are highly expressed in the LS (see below). Previous studies have shown that stress-induced HPA axis activity is regulated by a glucocorticoid-driven feedback inhibition that is mediated at least in part by limbic forebrain structures [START_REF] Herman | Limbic system mechanisms of stress regulation: Hypothalamo-pituitary-adrenocortical axis[END_REF]. Since both glucocorticoid and mineralocorticoid receptors are expressed in the LS [START_REF] Arriza | The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response[END_REF][START_REF] Ahima | Charting of type II glucocorticoid receptor-like immunoreactivity in the rat central nervous system[END_REF][START_REF] Ahima | Type I corticosteroid receptor-like immunoreactivity in the rat CNS: distribution and regulation by corticosteroids[END_REF][START_REF] Cintra | Mapping and computer assisted morphometry and microdensitometry of glucocorticoid receptor immunoreactive neurons and glial cells in the rat central nervous system[END_REF] this area might be a potential target for mediating glucocorticoid effects. However, neither injecting the selective glucocorticoid receptor antagonist mifepristone nor the selective mineralocorticoid receptor antagonist RU-28318 into the LS changed ACTH responses to forced swimming suggesting that this LS HPA-inhibitory mechanism is independent of a glucocorticoid mediated negative feedback mechanism. Thus, our data provide further evidence that the HPA axis can also be regulated by glucocorticoidindependent inhibition from neuronal sources. This is in line with findings from adrenalectomized animals that still can inhibit ACTH stress responses although lacking glucocorticoid feedback signals [START_REF] Jacobson | Circadian variations in plasma corticosterone permit normal termination of adrenocorticotropin responses to stress[END_REF].

Role of LS neurons on behavioral stress response

In addition to HPA axis regulation we found that the LS modulates stress coping behavior in the forced swim test, a widely used test to assess antidepressant properties of drugs. As LS-lesioned animals showed a more passive coping style which is often interpreted as behavioral despair [START_REF] Porsolt | Rodent models of depression: forced swimming and tail suspension behavioral despair tests in rats and mice[END_REF] our finding identifies the LS as a key brain area especially important in mediating active stresscoping strategies. Thus, our data strengthen the suggestion that the septum and especially the LS belongs to a "behavioral integration system" responsible for promoting the expression of relevant active behavioral responses in aversive and stressful situations (De [START_REF] Oca | Amygdala and periaqueductal gray lesions only partially attenuate unconditional defensive responses in rats exposed to a cat[END_REF][START_REF] Sheehan | Regulation of affect by the lateral septum: implications for neuropsychiatry[END_REF].

Interestingly, previous studies have shown that LS-lesions in rats produced an anxiogenic-like phenotype [START_REF] Yadin | The role of the lateral septum in anxiolysis[END_REF]. Accordingly, this effect was defined as "septal rage syndrome" -representing a generalized disinhibition of fear [START_REF] Brady | Subcortical mechanisms in emotional behavior: affective changes following septal forebrain lesions in the albino rat[END_REF][START_REF] Albert | The septal forebrain and the inhibitory modulation of attack and defense in the rat. A review[END_REF][START_REF] Sheehan | Regulation of affect by the lateral septum: implications for neuropsychiatry[END_REF]. Furthermore, septal damage has been found to be correlated with an increase in the expression of defensive behaviors, which is suggestive of an increase in fear [START_REF] Sparks | Septal lesions potentiate freezing behavior to contextual but not to phasic conditioned stimuli in rats[END_REF]. However, different LS lesion studies suggest some anxiolytic-like effects in the elevated plus-maze and shock-probe burying test after LS ablation [START_REF] Pesold | Excitotoxic lesions of the septum produce anxiolytic effects in the elevated plus-maze and the shock-probe burying tests[END_REF][START_REF] Menard | Lateral and medial septal lesions reduce anxiety in the plusmaze and probe-burying tests[END_REF]. Thus, the exact role of the LS in modulation of anxiety and emotion-related behaviors is still not fully understood. Methodological differences such as extent of lesions and ways of application might be responsible for the disparity between findings. It might be speculated that even subregions of the LS mediate different and distinct behavioral functions and therefore it depends on the exact position and extent of lesions which effects are finally produced.

Interestingly, previous studies point to a critical role of the LS in mediating behavioral symptoms of depression. For instance, in the learned helplessness paradigm in rats, a widely used animal model of depression, it has been shown that helpless animals show lower stress-induced neuronal activation within the LS than controls [START_REF] Steciuk | Decrease in stress-induced c-Fos-like immunoreactivity in the lateral septal nucleus of learned helpless rats[END_REF]. However, stress resistant rats which received the same inescapable shock but did not become helpless showed no reduction in septal activity indicating that a normal functioning of the LS is required for effective coping responses to inescapable stress. Conversely, substantial evidence suggests that antidepressants ameliorate the behavioral manifestations of depression by restoring the neuronal activity in the LS [START_REF] Contreras | Action of antidepressants on the septal nuclei of the rat[END_REF][START_REF] Contreras | Chronic clomipramine increases firing rate in lateral septal nuclei of the rat[END_REF][START_REF] Contreras | The lowest effective dose of fluoxetine in the forced swim test significantly affects the firing rate of lateral septal nucleus neurones in the rat[END_REF][START_REF] Lino-De-Oliveira | Effects of acute and chronic fluoxetine treatments on restraint stress-induced Fos expression[END_REF][START_REF] Muigg | Altered brain activation pattern associated with drug-induced attenuation of enhanced depression-like behavior in rats bred for high anxiety[END_REF]. Therefore, we injected mifepristone, a selective glucocorticoid receptor antagonist with anxiolytic and antidepressant properties (De [START_REF] Kloet | Antiglucocorticoid RU 38486 attenuates retention of a behaviour and disinhibits the hypothalamic-pituitary adrenal axis at different brain sites[END_REF][START_REF] Korte | Anxiolytic-like effects of selective mineralocorticoid and glucocorticoid antagonists on fear-enhanced behavior in the elevated plus-maze[END_REF][START_REF] Debattista | The use of mifepristone in the treatment of neuropsychiatric disorders[END_REF][START_REF] Schatzberg | Glucocorticoid antagonists in neuropsychiatric [corrected] disorders[END_REF][START_REF] Wulsin | Mifepristone decreases depression-like behavior and modulates neuroendocrine and central hypothalamic-pituitaryadrenocortical axis responsiveness to stress[END_REF] locally into the LS and measured behavioral stress coping behavior in the forced swim test. Notably, we found that local administration of mifepristone into the LS increased active coping and reduced floating behavior indicating an antidepressant-like effect. This behavioral effect seems to be mediated by glucocorticoid receptors since intraseptal administration of a mineralocorticoid receptor antagonist had no effect. Thus, our data extend previous findings demonstrating that intraseptal injection of mifepristone normalizes the anxiogenic response in the elevated plus-maze test induced by either previous social defeat or systemic corticosterone administration [START_REF] Calfa | Glucocorticoid receptors in lateral septum are involved in the modulation of the emotional sequelae induced by social defeat[END_REF]. However, mifepristone is not an ideal glucocorticoid-receptor antagonist, because it has also some affinity to progesterone receptors. Since progesterone receptors are also expressed in the lateral septum [START_REF] Maclusky | Progestin receptors in rat brain: distribution and properties of cytoplasmic progestin-binding sites[END_REF][START_REF] Sar | Immunohistochemical localization of estrogen receptor in rat brain, pituitary and uterus with monoclonal antibodies[END_REF]) and intraseptally administered progesterone has been shown to influence forced swim behavior [START_REF] Estrada-Camarena | Participation of the lateral septal nuclei (LSN) in the antidepressant-like actions of progesterone in the forced swimming test (FST)[END_REF], it is possible that these receptors are involved in the mediation of the behavioral effects of mifepristone. Further studies with more specific ligands for the glucocorticoid receptor are required to exclude this possibility.

Involvement of septal 5-HT 1A receptors in stress responses

After identifying a stress-inhibitory role of the LS we wanted to gain insight into the neurochemistry involved this effect. Previous studies suggest that the septal 5-HT system plays an important role in mediating behavioral and physiological responses to emotionally significant events. Both a dense serotonergic innervation of the LS from the raphe nuclei [START_REF] Köhler | The distribution and origin of serotonin-containing fibers in the septal area: a combined immunohistochemical and fluorescent retrograde tracing study in the rat[END_REF] and various 5-HT receptors primarily of the 5-HT 1A subtype are highly expressed in the LS [START_REF] Kia | Immunocytochemical localization of serotonin1A receptors in the rat central nervous system[END_REF][START_REF] Lanfumey | Central 5-HT(1A) receptors: regional distribution and functional characteristics[END_REF]. Therefore, we bidirectonally modulated septal 5-HT 1A receptors by administration of selective 5-HT 1A receptor agonist and antagonists into the LS and examined the role of this receptor on neuroendocrine and behavioral stress responses. We found a suppression of the stress-induced ACTH levels after 5-HT 1A receptor agonist administration and an increased and prolonged ACTH response after 5-HT 1A receptor blockade. Thus, our data suggest an inhibitory role of endogenous 5-HT within the LS on stress-induced HPA axis activation via intraseptal 5-HT 1A receptors. Notably, blockade of septal 5-HT 1A receptors by WAY-100635 mimics lesion effects while activation of these receptors facilitates the inhibitory role of the LS. The exact mechanism, however, mediating this inhibitory effect to the PVN remains unclear. It was shown previously that LS 5-HT 1A receptors were located primarily on calbindin-positive neurons indicative for septal GABAergic interneurons [START_REF] Aznar | The 5-HT1A serotonin receptor is located on calbindin-and parvalbumin-containing neurons in the rat brain[END_REF][START_REF] Lüttgen | 5-HT1A receptor mRNA and immunoreactivity in the rat medial septum/diagonal band of Brocarelationships to GABAergic and cholinergic neurons[END_REF]. Moreover, electrophysiological experiments have established that septal 5-HT 1A receptor activation causes neuronal hyperpolarisation [START_REF] Joels | Effect of serotonin and serotonin analogues on passive membrane properties of lateral septal neurons in vitro[END_REF][START_REF] Van Den Hooff | Actions of 5-hydroxytryptamine and 5-HT1A receptor ligands on rat dorso-lateral septal neurones in vitro[END_REF]. Thus, it is conceivable that a reduced 5-HT function in the LS either during psychological stress exposure [START_REF] Kirby | Regional differences in the effects of forced swimming on extracellular levels of 5-hydroxytryptamine and 5hydroxyindoleacetic acid[END_REF][START_REF] Ebner | Neurokinin 1 receptor antagonism promotes active stress coping via enhanced septal 5-HT transmission[END_REF] or after 5-HT 1A receptor blockade is associated with an enhanced intraseptal inhibitory function (presumably via GABAergic interneurons). Since a major part of LS output neurons are GABAergic [START_REF] Gallagher | Activities of neurons within the rat dorsolateral septal nucleus (DLSN)[END_REF][START_REF] Risold | Connections of the rat lateral septal complex[END_REF] and given that several LS projections to the PVN are GABAergic this GABA-to-GABA pathway would result in disinhibition of HPA axis stress responses. However, it is also conceivable that increased HPA axis stress responses are mediated not directly through GABAergic efferents to the PVN but indirectly through glutamatergic projections to inhibitory PVN projecting regions, including the peri-PVN region [START_REF] Herman | Role of GABA and glutamate circuitry in hypothalamo-pituitary-adrenocortical stress integration[END_REF][START_REF] Cullinan | Functional role of local GABAergic influences on the HPA axis[END_REF]. Both scenarios would lead to enhanced stress hormone levels during forced swimming resulting from a decreased inhibitory action of the LS on the PVN. Further studies using tract tracing methods combined with immunohistochemical techniques (e.g. c-Fos, GABA) should clarify the exact pathways and mechanisms underlying this septal HPA axis regulatory mechanism.

Interestingly, the suppression of stress-induced ACTH levels after intraseptal 5-HT 1A receptor activation was accompanied by behavioral alterations during forced swimming, including reduction of immobility and an increase in active coping (e.g. struggling, swimming). Thus, our data confirm earlier studies also demonstrating antidepressant-like effects after intraseptal 8-OH-DPAT administration in the classical forced swim test [START_REF] Schreiber | Neuroanatomical basis for the antidepressant-like effects of the 5-HT(1A) receptor agonists 8-OH-DPAT and ipsapirone in the rat forced swimming test[END_REF] and learned helplessness model [START_REF] Martin | Antidepressant-like action of 8-OH-DPAT, a 5-HT1A agonist, in the learned helplessness paradigm: evidence for a postsynaptic mechanism[END_REF]. In the present study, we used the modified version of the forced swim test [START_REF] Cryan | Assessing substrates underlying the behavioral effects of antidepressants using the modified rat forced swimming test[END_REF] to assess the rodent's reactivity to the novel stress environment without any potential confounds of learning/memory processes associated with the pretest session performed in the traditional test. These modifications make the test more sensitive for serotonergic antidepressant drugs such as SSRIs [START_REF] Detke | Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants[END_REF][START_REF] Cryan | Assessing substrates underlying the behavioral effects of antidepressants using the modified rat forced swimming test[END_REF]. Indeed, preliminary data from our lab shows a reduction of floating behavior and an enhancement of struggling behavior in our modified forced swim test after intraseptal administration of citalopram (Ebner et al, unpublished data). Interestingly, we could not observe any behavioral changes during the forced swim session in animals that were intraseptally infused with the selective 5-HT 1A receptor anatgonist WAY-100635. This might be surprising in respect to the observed effects of WAY-100635 in modulating the neuroendocrine stress response. However, although the reason for the behavioral ineffectiveness is currently unknown, it could be related to the low endogenous 5-HT tonus in the LS during forced swimming [START_REF] Kirby | Regional differences in the effects of forced swimming on extracellular levels of 5-hydroxytryptamine and 5hydroxyindoleacetic acid[END_REF][START_REF] Ebner | Neurokinin 1 receptor antagonism promotes active stress coping via enhanced septal 5-HT transmission[END_REF]. Therefore, it is conceivable that under those conditions the endogenous 5-HT level is not sufficient for significant behavioral effects after 5-HT 1A receptor blockade. Indeed, the lack of a behavioral effect in the forced swim test after 5-HT 1A receptor blockade was also reported after systemic administration [START_REF] Vry | 5-HT1A receptor agonists: recent developments and controversial issues[END_REF][START_REF] Vry | 5-HT1A receptors are differentially involved in the anxiolytic-and antidepressant-like effects of 8-OH-DPAT and fluoxetine in the rat[END_REF][START_REF] Cryan | Assessing substrates underlying the behavioral effects of antidepressants using the modified rat forced swimming test[END_REF].

Taken together, our results show that the LS plays an important role in promoting a HPA-axis inhibitory mechanism and active coping strategies during stress exposure. Furthermore, we found that this HPA inhibitory mechanism of the LS acts independent from glucocorticoid mediated negative feedback mechanisms. However, our data indicate a critical role of septal 5-HT 1A receptors in mediating stresspreventing actions of the LS as activation of septal 5-HT 1A receptors inhibits neuroendocrine and facilitates behavioral stress responses. Thus, our study supports a model in which enhanced activity of LS neurons is required for effective stress coping responses which provides improved protection from the deleterious effects of exaggerated psychological stress exposure.

Disclosure/Conflict of Interest

The author(s) declare that, except for income received from my primary employer, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.

Figure legends

Fig. 1 .

 1 Fig. 1. Placement and extent of bilateral LS-lesions. A) Schematic drawing of a

Fig. 2 .

 2 Fig. 2. Schematic drawing and representative microphotographs of cresyl-stained

Fig. 3 .

 3 Fig. 3. Effects of LS-lesions on HPA axis responses to forced swimming. A) Plasma

Fig. 4 .

 4 Fig. 4. Effects of LS-lesions on stress coping parameters during a 5-min forced

Fig. 5 .

 5 Fig. 5. Effects of intraseptal administration of the glucocorticoid receptor antagonist

Fig. 6 .

 6 Fig. 6. Effects of intraseptal administration of the 5-HT 1A receptor agonist 8-OH-DPAT (10 µg/side; black arrow) on neuroendocrine and behavioral stress response.

Fig. 7 .

 7 Fig. 7. Effects of intraseptal infusion of the 5-HT 1A receptor antagonist WAY-100635 (10µM) via retrodialysis (black bar) on basal and swim stress-induced ACTH (A) or

Acknowledgments:

This work was supported by the Austrian Science Fund FWF (K.E.).