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Molecular Endocrinology, doi:10.1210/me.2005-0262
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Molecular Endocrinology 20 (1): 219-231
Copyright © 2006 by The Endocrine Society

Impaired Reproductive Behavior by Lack of GluR-B Containing AMPA Receptors But Not of NMDA Receptors in Hypothalamic and Septal Neurons

Derya R. Shimshek, Thorsten Bus, Valery Grinevich, Frank N. Single, Volker Mack, Rolf Sprengel, Daniel J. Spergel1 and Peter H. Seeburg1

Department of Molecular Neuroscience, Max-Planck-Institute for Medical Research, 69120 Heidelberg, Germany

Address all correspondence and requests for reprints to: Daniel J. Spergel, Section of Endocrinology, Department of Pediatrics, University of Chicago, 5841 South Maryland Avenue, MC 5053, Chicago, Illinois 60637-1470. E-mail: dspergel{at}peds.bsd.uchicago.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The roles of ionotropic glutamate receptors in mammalian reproduction are unknown. We therefore generated mice lacking a major subtype of (S)-{alpha}-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA) receptors or all N-methyl-D-aspartate (NMDA) receptors in GnRH neurons and other mainly limbic system neurons, primarily in hypothalamic and septal areas. Male mice without NMDA receptors in these neurons were not impaired in breeding and exhibited similar GnRH secretion as control littermates. However, male mice lacking GluR-B containing AMPA receptors in these neurons were poor breeders and severely impaired in reproductive behaviors such as aggression and mounting. Testis and sperm morphology, testis weight, and serum testosterone levels, as well as GnRH secretion, were unchanged. Contact with female cage bedding failed to elicit male sexual behavior in these mice, unlike in control male littermates. Their female counterparts had unchanged ovarian morphology, had bred successfully, and had normal litter sizes but exhibited pronounced impairments in maternal behaviors such as pup retrieval and maternal aggression. Our results suggest that NMDA receptors and GluR-B containing AMPA receptors are not essential for fertility, but that GluR-B containing AMPA receptors are essential for male and female reproduction-related behaviors, perhaps by mediating responses to pheromones or odorants.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PULSATILE SECRETION of GnRH (GnRH, GnRH-I, or LHRH) from hypothalamic GnRH neurons, which is required for gonadal development and reproduction, is modulated by a variety of neurotransmitters including glutamate (1). Glutamate is the main excitatory neurotransmitter in the brain, including the neuroendocrine system (2), and its effects are mediated by the (S)-{alpha}-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) classes of glutamate receptor, as well as by kainate and metabotropic glutamate receptors. Using electrophysiological recording of glutamate-evoked currents in GnRH neuronal somata of GnRH-GFP transgenic mice in which GnRH neurons are marked with GnRH promoter-driven green fluorescent protein (GFP), we and others demonstrated that GnRH neurons express functional AMPA receptors (AMPARs) and NMDA receptors (NMDARs) (3, 4). Electron microscopic findings revealing that vesicular glutamate transporter 2 (VGluT2)-immunoreactive nerve terminals establish asymmetric synaptic contacts on GnRH neuronal dendrites and somata provided further evidence for direct glutamatergic innervation of GnRH neurons (5).

AMPARs are tetrameric ion channels consisting of combinations of GluR-A, -B, -C, and -D (or GluR1–4) subunits (6). The GluR-B subunit, prominently expressed in most excitatory neurons, is RNA edited at the glutamine/arginine site and hence confers a linear current-voltage relationship and low Ca2+ permeability to the AMPA channel (7). The linear current-voltage relationship of AMPA channel activity observed in GFP-expressing GnRH neurons (3) indicates that AMPA channels in GnRH neurons contain the glutamine/arginine site-edited GluR-B subunit. In GluR-B knockout mice, AMPA channels exhibit an inwardly rectifying current-voltage relationship and high Ca2+ permeability (8). Although GluR-B knockout mice display enhanced long-term potentiation, increased mortality, reduced exploration, and impaired motor coordination (8), the role of the GluR-B subunit in reproduction has not been investigated.

NMDARs are tetrameric ion channels consisting of the principal NR1 subunit and modulatory NR2A, NR2B, NR2C, or NR2D (or NMDAR1 and NMDAR2A-D) subunits (6). In our previous recordings, approximately 20% of GFP-labeled GnRH neurons were seen to express NMDARs (3), raising the possibility that these receptors are relevant for the function of the GnRH neuronal network. Unfortunately, NR1 knockout mice, which lack all NMDARs, cannot be used to investigate the role of these receptors in reproduction because they die soon after birth (9).

The Cre-lox system permits spatially restricted deletion of genes of interest (10). Using transgenic mice in which a 3.5-kb fragment of the GnRH promoter drives the expression of a codon-improved Cre gene (iCre), we showed that Cre-mediated deletion of a floxed gene occurs in GnRH neurons and other hypothalamic and septal neurons in which the GnRH promoter has been active during development (11). In the present study, we employed GnRH-iCre mice carrying floxed alleles for GluR-B or NR1, as well as GnRH-GFP mice, to examine the roles in fertility and reproduction of GluR-B containing AMPARs and of NMDARs. Our results suggest that neither GluR-B nor NR1 expression in GnRH neurons is essential for fertility, but that GluR-B expression in other hypothalamic and septal neurons is critical for male and female reproduction-related behaviors.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Brain Areas with Cre Activity in GnRH-iCre Mice
A transgene for Cre recombinase under the control of a 3.5-kb mouse GnRH promoter (GnRH-iCre) is expressed in GnRH neurons as well as in other, mainly limbic system, neurons of adult GnRH-iCre mice. We demonstrated this previously using Cre immunostaining, which provides a snapshot of Cre expression at the time the mouse is killed (11). Intriguingly, as indicated by X-gal staining throughout the brains of adult, double transgenic offspring of GnRH-iCre and R26R (ROSA26 reporter) Cre indicator mice (12), in which Cre-mediated recombination enables transcription of the lacZ gene (Fig. 1Go, schematic diagram), the cumulative Cre activity pattern, which reflects the entire history of GnRH promoter and Cre activity before death, is more widespread (Fig. 1Go, blue staining). One group of the X-gal-stained areas (diagonal band of Broca, lateral preoptic area, medial preoptic area, most notably at the level of the organum vasculosum of the lamina terminalis, medial septal nucleus, and retrochiasmatic area) is known to contain GnRH and Cre-expressing neurons in the adult based on GnRH and Cre immunostaining (11, 13). A second group of the X-gal-stained areas (consisting of all of the first group of areas plus the lateral hypothalamic area, lateral septal nucleus, septofimbrial nucleus, and zona incerta) contains neurons that in the adult express Cre but not GnRH, similar to previous findings from GnRH promoter-driven lacZ mice (GnRH-lacZ) (14, 15, 16). A third group (including all of the first two groups of areas plus the anterior hypothalamic area, bed nucleus of the stria terminalis, caudate putamen, olfactory tubercle, and paraventricular thalamic nucleus) contains neurons that in the adult express neither Cre nor GnRH but were stained in some of the GnRH-lacZ mice (15), perhaps because X-gal staining and ß-galactosidase immunostaining are more sensitive than Cre immunostaining. A fourth group (all of the first three groups of areas plus the central amygdaloid nucleus, nucleus accumbens, posterior thalamic nucleus, paraventricular nuclear group, and reticular thalamus) contains neurons that in the adult express neither Cre nor GnRH and were not stained in GnRH-lacZ mice. In this last group, the GnRH promoter may have been active early in development only.



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  		Fig. 1. GnRH-iCre Transgene and Its Expression in GnRH-iCre/R26R Mice

Brain areas (abbreviations listed below) exhibiting Cre activity in a GnRH-iCre/R26R double transgenic mouse in which Cre, under the control of the GnRH promoter, excises the floxed phosphoglycerine kinase (PGK)-neomycin stop cassette 5' of the lacZ gene to permit lacZ transcription (schematic diagram) resulting in ß-galactosidase activity, indicated by X-gal staining (blue staining). ß-Galactosidase activity reflects Cre activity from the time the transgene first became active up until the time the mouse was killed for analysis. Tissue is counterstained red with eosin. Box indicates brain region shown in Fig. 2Go. Scale bars, 1 mm. The upper scale bar applies to the first brain section only, and the lower scale bar applies to all of the other sections. Acb, Nucleus accumbens; AHA, anterior hypothalamic area; BST, bed nucleus of the stria terminalis; Ce, central amygdaloid nucleus; CPu, caudate putamen; DBB, diagonal band of Broca; LH, lateral hypothalamic area; LPO, lateral preoptic area; LS, lateral septal nucleus; MPA, medial preoptic area; MS, medial septal nucleus; OVLT, organum vasculosum of the lamina terminalis; Po, posterior thalamic nuclear group; PV, paraventricular thalamic nucleus; RCh, retrochiasmatic area; Rt, reticular thalamus; Sfi, septofimbrial nucleus; Tu, olfactory tubercle; ZI, zona incerta.

 
We expected that all X-gal-stained brain areas in adult GnRH-iCre/R26R mice, regardless of the time during development at which the GnRH promoter and hence Cre were active, would lack GluR-B and NR1, respectively, in adult GluR-B{Delta}HS and NR1{Delta}HS mice (i.e. mice that were heterozygous for GnRH-iCre and homozygous for floxed GluR-B or NR1 produced by mating GnRH-iCre mice with mice containing a floxed allele for GluR-B or NR1; see schematic diagrams in Fig. 2AGo and B, respectively). Immunostaining for GluR-B (Fig. 2AGo) and NR1 (Fig. 2BGo) in adult (older than postnatal d 60, [> P60]) GluR-B{Delta}HS and NR1{Delta}HS mice confirmed this, although we did not check on a cell-to-cell basis. We note that the decrease in GluR-B and NR1 immunostaining in these mice was most pronounced in the hypothalamus and septal nuclei, where Cre activity (Fig. 1Go) appeared to be highest.



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  		Fig. 2. Cre-Mediated Ablation of GluR-B and NR1

GluR-B{Delta}HS (A) and NR1{Delta}HS (B) mice were generated by breeding GnRH-iCre mice with GluR-Bf and NR1f mice, respectively (schematic diagrams). A, Immunohistochemical detection of GluR-B in GluR-Bf/f (left) and GluR-B{Delta}HS mice (right). B, Immunohistochemical detection of NR1 in NR1f/f mice (left) and NR1{Delta}HS mice (right). The diagonal band of Broca (DBB), lateral septal nucleus (LS), and medial septal nucleus (MS) in the GluR-B{Delta}HS and NR1{Delta}HS mice are immunostained less intensely than in the GluR-Bf/f and NR1f/f mice, indicating reduced GluR-B and NR1 expression in those areas. The brain sections in A and B were from a brain level close to that of the third section (boxed) of Fig. 1Go. Scale bars, 200 µm.

 
Glutamate Responses of GnRH Neurons Lacking GluR-B or NR1
Currents measured from six of seven (~86%) fluorescing GnRH neurons of adult (> P60) GluR-B{Delta}HS/GnRH-GFP mice (n = 4) in response to glutamate were inwardly rectifying, with only a tiny outward current at a test potential of +20 mV, and smaller than in seven GFP-labeled GnRH neurons of control littermates (n = 4) (Fig. 3Go, A and B). These changes in property and amplitude of AMPA currents are consistent with our earlier finding that Cre is expressed and/or active in approximately 93% of GnRH neurons in GnRH-iCre mice (11). Four of these seven GnRH neurons exhibited an NMDA response (data not shown). These findings suggest that most GnRH neurons of GluR-B{Delta}HS mice express AMPARs lacking GluR-B required for a linear current-voltage relationship, and that expression of GluR-A, -C, or -D subunits, or NMDARs, is not altered upon GluR-B deletion.



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  		Fig. 3. GluR-B{Delta}HS and NR1{Delta}HS Mice Exhibit Altered Responses to Glutamate

A, Glutamate (Glu)-evoked currents in a nucleated patch of a GFP-expressing GnRH neuron from a GluR-Bf/f/GnRH-GFP (control) mouse (left traces) and a GluR-B{Delta}HS/GnRH-GFP (mutant) mouse (right traces). Glutamate (1 mM) was applied every 5 sec for 50 ms at holding potentials (Vh’s) of –100, –80, –60, –40, –20, 0, 20, 40, 60, 80, and 100 mV. An arrow points to each response. In this and subsequent traces, patches were initially held at –60 mV and then stepped to a new Vh before agonist application. B, I-V relationships of the averaged responses to glutamate in controls and mutants (n = 7 GnRH neurons in each group). C, Glutamate (Glu; top traces) and NMDA (bottom traces)-evoked current in a nucleated patch of a GFP-expressing GnRH neuron from an NR1f/f/GnRH-GFP (control) mouse (left traces) and an NR1{Delta}HS/GnRH-GFP (mutant) mouse (right traces). The AMPA component of the response to glutamate remains despite the absence of the NMDA component. D, Percentage of GFP-expressing GnRH neurons of control and mutant mice responding to NMDA or having an NMDA component in their response to glutamate. Asterisk indicates a significant difference (P < 0.05) between controls and mutants. The numbers of control and mutant GnRH neurons recorded from are indicated in parentheses.

 
In all ten GFP-labeled GnRH neurons of NR1{Delta}HS/GnRH-GFP mice examined (n = 2), the fast-activating and -inactivating AMPA component of the response after glutamate application was present but the slowly activating and deactivating NMDA component of the response, and the response to NMDA application, were absent (Fig. 3Go, C and D). This result indicated that the GnRH neurons lacked the NR1 subunit and, therefore, functional NMDARs and was in contrast to control littermates (n = 13), in which 12 of 27 (~44%) GnRH neurons exhibited an NMDA component in their glutamate response, or responded to NMDA (P < 0.05, mutants vs. controls). Collectively, these results provided further evidence that transgenic Cre expression in our mice ablated functional GluR-B containing AMPARs and NMDARs in GnRH and other hypothalamic as well as septal neurons.

Testicular and Sperm Morphology, Testes Weight, Serum Testosterone, and GnRH Secretion in Male GluR-B{Delta}HS Mice
To determine whether GluR-B ablation affected fertility in male GluR-B{Delta}HS mice, we analyzed testicular morphology, isolated sperm, and measured testes weight and serum testosterone levels of GluR-B{Delta}HS mice, all of which depend on GnRH secretion from GnRH neurons. At the light microscopic level, the testes of GluR-B{Delta}HS mice (n = 4 mice) were morphologically indistinguishable from those of control (GluR-Bf/f) littermates (n = 4 mice; Fig. 4AGo). Although there were large variations in the sizes of the seminiferous tubules in GluR-B{Delta}HS mice and their control littermates, overall testicular morphology and the sizes of the spermatogonia, spermatids, and spermatozoa (Fig. 4AGo), as well as the size (Fig. 4BGo) and motility (n = 3; data not shown) of spermatozoa contained in semen in GluR-B{Delta}HS mice were comparable to those in control littermates. Testes weights (expressed as grams of tissue wet weight; combined weight of both testes) in GluR-B{Delta}HS mice (0.210 ± 0.12 g; n = 13 mice) were also similar (P > 0.05) to those of control littermates (0.241 ± 0.014 g; n = 12 mice). Moreover, serum testosterone levels of male GluR-B{Delta}HS mice (3.83 ± 2.02 ng/ml; n = 6 mice) did not differ significantly (P > 0.05) from those of controls (3.94 ± 1.58 ng/ml; n = 10 mice). The comparable serum testosterone levels in male GluR-B{Delta}HS and control mice predict unaltered GnRH secretion, which is required for LH and FSH release from the anterior pituitary and subsequent secretion of testosterone from the testis.



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  		Fig. 4. Testicular and Sperm Morphology Are Similar in GluR-B{Delta}HS Mice to that in Control Littermates but Male GluR-B{Delta}HS Mice Are Comparatively Poor Breeders and Exhibit Dramatically Impaired Mounting and Aggression

A, Testis morphology. Low (left) and high (right) magnification views of a 1-µm-thick section of testis from a control (top) and a GluR-B{Delta}HS (bottom) mouse showing various stages of spermatogenesis. sg, Spermatogonia; scI, primary spermatocytes; scII, secondary spermatocytes; st, spermatids; sz, spermatozoids. B, Sperm from a control (top) and a GluR-B{Delta}HS (bottom) mouse. Scale bars in A and B, 100 µm. C, Male breeding success, expressed as the percentage of male GluR-B{Delta}HS, NR1{Delta}HS, and control (GluR-Bf/f) mice that generate offspring. D, Mounting and aggression of male GluR-B{Delta}HS, NR1{Delta}HS, and control (GluR-Bf/f) mice. In C and D, the numbers of mice tested are indicated in parentheses and significant differences (P < 0.05) between groups are denoted by asterisks. The breeding success, mounting, and aggression of male GluR-B{Delta} mice are shown for comparison.

 
Indeed, as determined from hypothalamic slices, mean GnRH secretion, GnRH pulse amplitude, and GnRH pulse frequency in GluR-B{Delta}HS (4.70 ± 0.72 pg/ml, 1.92 ± 0.30 pg/ml, and 0.40 ± 0.07 pulses/h, respectively; n = 8 mice) and NR1{Delta}HS (4.01 ± 0.52 pg/ml, 1.21 ± 0 pg/ml, and 0.43 ± 0.14 pulses/h, respectively; n = 3 mice) males were unchanged (P > 0.05) when compared with control (GluR-Bf/f) males (3.55 ± 0.42 pg/ml, 1.37 ± 0.14 pg/ml, and 0.44 ± 0.11 pulses/h, respectively; n = 8 mice). The values for GnRH pulse amplitude (< 2 pg/ml) and frequency (< 1 pulse/2 h) were lower than those previously reported in a variety of preparations (17, 18, 19, 20, 21, 22). However, the slices appeared to remain viable throughout each experiment because in response to depolarization with Ringer solution containing 37.5 mM KCl at the end of each experiment they exhibited GnRH secretory responses (expressed as peak minus baseline secretion), of 2.65 ± 1.02 pg/ml (GluR-B{Delta}HS; n = 8 mice), 3.83 ± 1.13 pg/ml (NR1{Delta}HS; n = 3 mice), and 1.87 ± 0.20 pg/ml (GluR-Bf/f, n = 8 mice).

Hence, testicular and sperm morphology, testosterone levels, and GnRH secretion were unaltered in male GluR-B{Delta}HS mice.

Breeding Performance of Male GluR-B{Delta}HS and NR1{Delta}HS Mice
To evaluate whether the lack of GluR-B or NR1 in the Cre-expressing neurons of our mice would have any impact on reproduction despite having no apparent effect on fertility, we examined the breeding performance of male GluR-B{Delta}HS and NR1{Delta}HS mice. Figure 4CGo shows that compared with control (male GluR-Bf/f) littermates and male NR1{Delta}HS mice, which almost always produced offspring when mated with wild-type mice, male GluR-B{Delta}HS mice rarely produced offspring (P < 0.05). Not surprisingly, male GluR-B{Delta} mice, in which GluR-B is deleted in the same brain areas as in GluR-B{Delta}HS mice as well as in all other brain areas in which GluR-B is normally expressed, were also unsuccessful at breeding. Thus, breeding performances of male NR1{Delta}HS mice were not affected, whereas male GluR-B{Delta}HS mice showed dramatic impairments in reproduction.

Mounting and Aggression of Male GluR-B{Delta}HS Mice
To determine whether the impaired breeding performance of male GluR-B{Delta}HS mice derived from altered reproductive behavior, we examined mounting and aggression, and saw significant differences compared with controls (P < 0.05) (Fig. 4DGo). Control male littermates mounted female mice several times during the night, whereas neither GluR-B{Delta}HS nor GluR-B{Delta} males ever showed interest in the female control mice. Moreover, control male, but not GluR-B{Delta}HS or GluR-B{Delta} male, host mice attacked a wild-type male intruder during 10-min test periods.

Ovarian Morphology of Female GluR-B{Delta}HS Mice and Breeding Performance, Pup Retrieval, and Maternal Aggression of Female GluR-B{Delta}HS and NR1{Delta}HS Mice
We also examined the ovarian morphology and breeding performance of female GluR-B{Delta}HS mice, and the reproduction-related behaviors of female GluR-B{Delta}HS and NR1{Delta}HS mice. Figure 5AGo shows that ovarian morphology, including the appearance of corpora lutea, in female GluR-B{Delta}HS mice did not differ from that in control female littermates. Figure 5BGo shows that although fewer female GluR-B{Delta}HS and GluR-B{Delta} mice produced offspring than control (female GluR-Bf/f) or female NR1{Delta}HS littermates, the difference was not statistically significant (P > 0.05). Those female GluR-B{Delta}HS mice that did produce offspring had normal litter sizes (8 ± 1 pups; n = 8 GluR-B{Delta}HS mothers; control littermate litter size: 9 ± 1 pups; n = 15 control littermate mothers). Thus, the fertility and breeding performance of female GluR-B{Delta}HS and NR1{Delta}HS mice were not affected.



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  		Fig. 5. Similar Ovarian Morphology and Breeding Performance, but Dramatically Impaired Pup Retrieval and Maternal Aggression in Female GluR-B{Delta}HS Mice Compared with NR1{Delta}HS and Control (GluR-Bf/f) Mice

A, Ovarian morphology in 3-month-old female GluR-B{Delta}HS mice (n = 3) and aged-matched control (GluR-Bf/f) littermates (n = 2). Low (left) and high (right) magnification views of a 1-µm-thick section of ovary from a control (top) mouse and a GluR-B{Delta}HS (bottom) mouse. Scale bars, 100 µm. a, Atretic follicle; af, antral follicle; cl, corpus luteum; g, granulosa cell; o, oocyte; pf, primary follicle. B, Female breeding success, expressed as the percentage of female GluR-B{Delta}HS, NR1{Delta}HS, and control (GluR-Bf/f) mice that generate offspring. C, Pup retrieval and maternal aggression of female GluR-B{Delta}HS, NR1{Delta}HS, and control (GluR-Bf/f) mice. In B and C, the numbers of mice tested are indicated in parentheses, and significant differences (P < 0.05) between groups are denoted by asterisks. The breeding success, pup retrieval, and maternal aggression of female GluR-B{Delta} mice are shown for comparison.

 
We also examined the pup retrieval and maternal aggression behavior of female GluR-B{Delta}HS mice to ascertain whether impairments in male behavior due to GluR-B ablation also extended to female-typical behavior linked to reproduction. Female GluR-B{Delta}HS mice suckled their pups but exhibited impaired pup retrieval and maternal aggression compared with control female littermates (P < 0.05; Fig. 5CGo). Whereas female control mice retrieved their pups within the 5-min test period for pup retrieval and showed aggressive behavior toward male intruders within the 10-min test period, female GluR-B{Delta}HS mice failed to retrieve their pups and displayed no aggression within those periods. However, after 20–30 min, female GluR-B{Delta}HS mice built a new nest in the corner where the pups were placed for the retrieval test. Together with the failure to retrieve their pups, building the new nest suggested that the female GluR-B{Delta}HS mice had problems associating the pups with their original nest. Thus, female GluR-B{Delta}HS mice, like their male counterparts, exhibited pronounced impairments in reproduction-related behaviors.

Responses of Male GluR-B{Delta}HS Mice to Cage Bedding Containing Female Urine
The severe impairments of male GluR-B{Delta}HS mice in reproduction-related behavior could depend on the detection of and/or reaction to female pheromones/odorants. This we investigated by monitoring in an open field test the time spent in cage bedding containing female mouse urine. We found that compared with male control littermates (n = 8) male GluR-B{Delta}HS mice (n = 8) spent a similar amount of time in the middle compartment of the open field with clean bedding but significantly less time in cage bedding containing female mouse urine (P < 0.05; Fig. 6AGo). However, GluR-B{Delta}HS mice like their littermate controls were able to sense female pheromones/odorants because the time spent in the middle compartment was significantly increased (P < 0.05) when containing female bedding compared with clean bedding.



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  		Fig. 6. Reduced Responsiveness to Female Pheromones/Odorants of Male GluR-B{Delta}HS Mice Compared with Control Littermates in an Open Field Test

A, Male GluR-B{Delta}HS mice (n = 8) spend a similar amount of time (P > 0.05; n.s., no significant difference) as control littermates (GluR-Bf/f mice; n = 8) in clean cage bedding but significantly less time (P < 0.05) than control littermates in cage bedding soiled with female urine (i.e. female bedding). The time spent by male GluR-B{Delta}HS and control littermate mice in female bedding was significantly longer (P < 0.05) than that spent in clean bedding before (left) and after (right). B, Distance traveled in each open field trial was not different (P > 0.05) in male GluR-B{Delta}HS mice compared with control littermates.

 
Interestingly, unlike for littermate controls, the female bedding did not stimulate any sexual behavior in male GluR-B{Delta}HS mice, like digging in and searching through female bedding (seven of eight mice; see supplemental video published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Note that the overall distance traveled during the open field tasks did not differ between male control littermates and GluR-B{Delta}HS males (Fig. 6BGo). Hence, GluR-B appears to be required for reproduction-related behavior by participating in the response to female pheromones/odorants.

Body Weight, Grip Strength, Exploratory Behavior, and Anxiety of Male GluR-B{Delta}HS Mice
We tested whether the impairments in male GluR-B{Delta}HS mice were confined to reproduction-related behavior or also affected other parameters, such as body weight, grip strength, exploratory behavior, and anxiety. We observed that male GluR-B{Delta}HS mice showed similar body weight (24.2 ± 1.2 g, n = 9, P > 0.05) and exploratory behavior in an open-field test (1442 ± 161 cm, n = 12, P > 0.05), but slightly reduced grip strength (1.4 ± 0.04 N, n = 15, P < 0.05) compared with control littermates (body weight: 24.8 ± 0.8 g, n = 5; open field exploration: 1629 ± 144 cm, n = 10; grip strength: 1.6 ± 0.09 N, n = 10). In a dark-light box anxiety test, male GluR-B{Delta}HS mice exhibited a reduced number of exits (4 ± 1, n = 9, P < 0.05) and an increase in the latency of first exit (114 ± 37 sec, n = 9, P < 0.05), but spent a similar time in the lit compartment (45 ± 14 sec, n = 9, P > 0.05), compared with control littermates (number of exits: 9 ± 1, n =10; latency of first exit: 24 ± 6 sec, n = 10; time spent in lit compartment: 69 ± 10 sec, n = 10). We note that none of these parameters was affected as severely as reproductive behavior.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Transgenic GnRH-iCre mice with improved Cre recombinase (iCre) expression driven by the GnRH gene promoter fragment we previously employed for transgenic GFP expression in GnRH neurons (GnRH-GFP mice) (3) have Cre activity in about 90% of GnRH neurons. Cre activity in the majority of GnRH-iCre mice also occurs in non-GnRH neurons in the hypothalamus and septum (11) as well as in a small number of other forebrain regions (present study). The widespread cumulative Cre activity in hypothalamic, septal, and a small number of other forebrain areas as indicated by the ROSA26 lacZ reporter (R26R) gene suggests that only a fraction of cells in the embryo in which the GnRH promoter and hence Cre are active from embryonic days onwards will end up as hypothalamic GnRH neurons (23, 24, 25) because only those cells continue to exhibit GnRH promoter activity and produce GnRH, consistent with published mouse GnRH promoter-driven transgene profiles (15, 16). Because GnRH promoter-driven Cre targeted almost all GnRH neurons and produced, in addition, a much more circumscribed gene ablation than global gene knockouts, we employed GnRH-iCre mice and floxed alleles for GluR-B and NR1 subunit genes to ablate GluR-B containing AMPARs and NMDARs in GnRH and the other Cre-expressing neurons. These genetic manipulations permitted us to investigate the role of the principal ionotropic glutamate receptors in fertility and reproduction.

The electrophysiological measurements in adult (> P60) mice showed indeed that GluR-B containing AMPARs and NMDARs were ablated in GnRH neurons of the appropriate genotypes, and we concluded that these major GluRs were also lacking in the other circumscribed Cre-expressing hypothalamic, septal, and forebrain populations. We note that we have been unable to detect GluR-B or NR1 in GnRH neurons of wild-type mice by immunohistochemistry, perhaps because of very low levels of GluR-B and NR1 in GnRH neurons, as indicated by the fact that the somatic glutamate-evoked AMPA and NMDA currents in GnRH neurons are roughly an order of magnitude lower than in neighboring non-GnRH neurons and two orders of magnitude lower than in hippocampal CA1 neurons (3, 26, 27). Therefore, we relied on electrophysiological measurements to detect the presence or absence of GluR-B and NR1.

A major finding of this study is that Cre-mediated ablation of GluR-B or NR1 in GnRH neurons, as well as in hypothalamic, septal, and other forebrain non-GnRH neurons that may be embryonically related to GnRH neurons (or that may turn on the same transcription factors for expression of another gene that happens to use, at a minimum, the same combination as the GnRH promoter) had no discernible effects on fertility. Adult GluR-B{Delta}HS and NR1{Delta}HS mice had similar ovarian, testicular, and sperm morphology, testes weights, serum testosterone levels, and GnRH secretion as controls. We note that, whereas it is possible that the LH surge capacity of female GluR-B{Delta}HS mice is attenuated, the attenuation is likely to be minor given their normal ovarian morphology and level of breeding success. Fertility in mice lacking GluR-B was unaffected despite the altered current-voltage relationship and the increased Ca2+-permeability of AMPA channels. More surprising was the lack of effect of NR1 deletion on fertility or reproductive behavior given that Cre-mediated ablation of NR1 would remove all NMDARs in GnRH and other Cre-expressing hypothalamic/septal neurons. This suggests that NMDARs in these neurons are not required for fertility or reproductive behavior. However, whereas the NR1{Delta}HS mice ultimately attain reproductive competence, we are investigating the possibility that they, or the GluR-B{Delta}HS mice, are delayed in their onset of puberty like prepubertal rats injected with NMDAR antagonists (28).

The lack of impairment of fertility in the GluR-B{Delta}HS and NR1{Delta}HS mice may be due to one of three possibilities. The first possibility is that not all GnRH neurons are targeted (~86% of GnRH neurons in the case of GluR-B and only ~44% of GnRH neurons in the case of NR1). However, especially in the case of GluR-B, we view this as the least likely possibility because although few GnRH neurons can maintain fertility (29), they are unlikely to produce the similar levels of GnRH secretion. The second possibility is that GluR-B containing AMPARs and NMDARs in GnRH neurons are not involved or important in the regulation of GnRH secretion or fertility. Whereas this possibility clearly pertains to GluR-B containing AMPARs, which are expressed in all GnRH neurons in control mice and deleted by Cre in approximately 86% of GnRH neurons in GluR-B{Delta}HS mice, it also pertains to NMDARs. Only approximately 44% of GnRH neurons in control mice exhibited a response to NMDA or an NMDA-like response to glutamate; however, no GnRH neurons in NR1{Delta}HS mice exhibited such a response. Without the response, GnRH secretion and fertility were normal, implying that NMDARs in GnRH neurons are not essential for GnRH secretion or fertility. Our results are consistent with, but do not prove, this possibility. The third possibility is that other receptors, probably not other subunits because the AMPA currents were smaller and NR1 is required for functional NMDARs, have compensated for the loss of GluR-B and NR1 in development in GluR-B{Delta}HS and NR1{Delta}HS mice. Previous studies show that AMPA and NMDA increase GnRH secretion in immortalized GnRH neurons (30) and evoke depolarizing currents in native GnRH neurons (3, 4). Based upon our results, the fact that receptor compensation may have occurred, and these previous studies, we conclude that although GluR-B containing AMPARs and NMDARs in native GnRH neurons may modulate GnRH secretion and therefore play an important role in fertility, they are not essential for GnRH secretion or fertility.

The other major finding of our study is that the Cre-induced lack of GluR-B in select hypothalamic, septal, and other forebrain populations severely affected reproduction-related behaviors. Despite the apparent lack of effect of GluR-B ablation on fertility and litter size, male and female GluR-B{Delta}HS mice exhibited impaired reproduction-related behaviors (i.e. mounting, male aggression, pup retrieval, and maternal aggression). Male GluR-B{Delta}HS mice were poor breeders, comparable to complete GluR-B knockout mice. Female GluR-B{Delta}HS mice, in contrast to their male counterparts, were not impaired in breeding, and those female GluR-B{Delta}HS mice that produced offspring had normal numbers of pups. Male GluR-B{Delta}HS mice, like complete GluR-B knockouts, were severely impaired in mounting wild-type females and in aggression toward wild-type intruder males. Physical fitness, evaluated by measuring forelimb grip strength and exploratory activity, was affected only moderately and hence could not account for these impairments. We did observe a higher level of anxiety in male GluR-B{Delta}HS mice than control littermates. Female GluR-B{Delta}HS mice were impaired in pup retrieval and maternal aggression.

Although GluR-B knockout mice have been previously reported to display enhanced long-term potentiation, increased mortality, reduced exploration, and impaired motor coordination (8), to our knowledge the present report is the first to show the importance of GluR-B for reproduction-related behaviors. In male mice, the hypothalamus (including the preoptic area) and septum have been shown to be involved in steroid-dependent reproductive behaviors such as ultrasonic vocalizations, female urine preference, urine marking, and mounting (31). In female mice, the accessory olfactory bulb, parts of the amygdala, and the hypothalamus have been shown to be involved in maternal aggression (32), but the detailed circuitry and signaling mechanisms for these behaviors have yet to be elucidated. Taken together, our results suggest that GluR-B expression in those brain areas in the GluR-B{Delta}HS mice in which GluR-B is depleted, which include the hypothalamus and septum, is essential for male and female reproduction-related behaviors.

The present report is also the first to provide evidence suggesting that GluR-B containing AMPARs mediate responses to pheromones or odorants that trigger male and female reproduction-related behaviors. Despite intact odor sensing, the lack of sexual-related behavior upon exposure to female pheromones/odorants in male GluR-B{Delta}HS mice indicates that these impairments are due to the absence of GluR-B in Cre-expressing GnRH or non-GnRH neurons in parts of the hypothalamus and/or septal nuclei that may use pheromonal and/or odorant information to trigger reproduction-related behaviors. [The vomeronasal organ appeared to lack GnRH promoter-driven Cre activity (data not shown).]

Interestingly, the requirement of the GluR-B subunit for reproductive competence is in marked contrast to that of the GluR-A subunit. Male GluR-A knockout mice, unlike male GluR-B knockout (GluR-B{Delta}) or male GluR-B{Delta}HS mice are not impaired in breeding performance (33), although they are impaired in male aggression (34), suggesting that both GluR-A and GluR-B may be essential for male aggression but that GluR-B plays a more important role in breeding performance. Male mice expressing unedited GluR-B(Q) (26, 35) in hypothalamic/septal areas are also not impaired in breeding performance (Shimshek, D. R., unpublished observation). This suggests that the impairments we observed in GluR-B{Delta}HS mice are not due to increased Ca2+ permeability because incorporation of unedited GluR-B(Q) in AMPARs also yields Ca2+-permeable AMPARs. Thus, the absence of any response to pheromones/odorants in reproduction-related behaviors appears to be due to other changes, such as AMPAR conductance, or altered AMPAR trafficking/assembly or signal transduction (36, 37, 38, 39, 40, 41).

In conclusion, the present results suggest that GluR-B in GnRH promoter-driven Cre-expressing neurons is not essential for fertility but is essential for male and female reproduction-related behaviors. In contrast, and perhaps unexpectedly, NMDARs in GnRH promoter-driven Cre-expressing neurons are not essential for fertility or reproduction-related behaviors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
GluR-B{Delta}HS and NR1{Delta}HS Mice
GluR-B{Delta}HS and NR1{Delta}HS mice (i.e. mice with a hypothalamic and septal deletion of GluR-B or NR1) were generated by breeding transgenic GnRH-iCre mice (Ref.11 ; Figs. 1Go and 2Go, A and B) with gene-targeted mice having floxed (i.e. alleles flanked by loxP sites) GluR-B (exon 11 floxed) or NR1 (exons 11–18 floxed) alleles (Fig. 2Go, A and B, left). Cre-mediated recombination in hypothalamic and septal neurons of mice heterozygous for the GnRH-iCre transgene and homozygous for the floxed receptor alleles produced GluR-B{Delta}HS (Fig. 2AGo, right) and NR1{Delta}HS (Fig. 2BGo, right) mice. Region-specific Cre expression was visualized in brain sections by immunohistochemistry (see below) for GluR-B and NR1 (documenting loss of these subunits), as well as by X-gal staining in mice heterozygous for GnRH-iCre and the R26R reporter locus (12) (see below). In a very small number of cases, we observed more widespread X-gal staining than that shown in Fig. 1Go, which may reflect epigenetic instability common in transgenic approaches.

Genotyping
Mice were genotyped by PCR of mouse-tail DNA with specific primers indicated below together with the approximate lengths of the amplified DNA fragments. GnRH-iCre: GnRH51 (5'-GAAGTACTCAACCTACCAACGGAAG-3'), iCre32 (5'-CACAGACAGGAGCATCTTCCAG-3'), 400 bp. GluR-B2lox: VM12 (5'-GCGTAAGCCTGTGAAATACCTG-3'), VM10 (5'-GTTGTCTAACAAGTTGTTGACC-3'); wild-type, 250 bp; mutant, 340 bp. NR12lox: N1ex18do1 (5'-CTGGGACTCAGCTGTGCTGG-3'), N1in18up1 (5'-AGGGGAGGCAACACTGTGGAC-3'); wild-type, 460 bp; mutant, 530 bp. R26R (Cre reporter mouse; 12): lac3' (5'-TTACCCGTAGGTAGTCACGCA-3'), lac5' (5'-TTACGATGCGCCCATCTACAC-3'), 500 bp. GnRH-GFP: GnRH51 (5'-GAAGTACTCAACCTACCAACGGAAG-3'), hGFP1 (5'-GCCATCCAGTTCCACGAGAATTGG-3'), 280 bp. GluR-B1lox (GluR-B{Delta}): VM10 (5'-GTTGTCTAACAAGTTGTTGACC-3'), VM17 (5'-GAATCATTGTTGACAGATTGCCAC-3')’ wild-type, 1180 bp; mutant, 320 bp. VM42 (5'-CATTGTGGATTCAAGTACAAGC-3'), VM43 (5'-GAGTCAGTCGGAATAGTTGGCC-3'); wild-type, 350 bp. Mice that were heterozygous for the GnRH-iCre transgene and homozygous for the GluR-B2lox or NR12lox allele (i.e. GluR-B{Delta}HS or NR1{Delta}HS), as well as control littermates that were positive for the GnRH-iCre transgene or homo- or heterozygous for the GluR-B2lox or NR12lox allele (control littermates, referred to in the text for easier representation as GluR-Bf/f and NR1f/f), were generated. Some of these mice were bred with GnRH-GFP mice (3) to produce offspring to identify GnRH neurons for electrophysiological recordings of AMPA and NMDA channel activity.

Use and Care of Mice
Experiments were performed in accordance with protocol 35-9185.81/35/97 approved by the Regierungspräsidium, Karlsruhe, Germany. Adult male (2–13 months old) and randomly cycling female (2–9 months old) GnRH-iCre/R26R, GluR-B{Delta}HS, GluR-B{Delta}HS/GnRH-GFP, NR1{Delta}HS, and NR1{Delta}HS/GnRH-GFP mice of strain C57BL/6 or of mixed C57BL/6 and NMRI strain, as well as control littermate mice of the same strain, with or without the GnRH-GFP transgene, were used. GluR-B{Delta} mice, in which GluR-B was functionally ablated globally, were also used. Mice were housed in a temperature (22 C)- and light (on 800-2000 h)-controlled room with ad libitum access to food and water.

Immunohistochemical and X-Gal Staining
Brain areas with Cre activity were revealed by breeding GnRH-iCre mice with R26R reporter mice (12) and performing X-gal staining on vibratome slices (100 µm thick) of GnRH-iCre/R26R mice (11). Immunohistochemistry for GluR-B or NR1 was performed on 4% paraformaldehyde/PBS-fixed brain slices (50–70 µm thick) of GluR-B{Delta}HS, NR1{Delta}HS mice, or control littermates, using rabbit polyclonal GluR-B (1:50; Chemicon International, Hofheim, Germany) or NR1 (1:100; Chemicon International) antiserum followed by horseradish peroxidase-coupled goat antirabbit IgG (1:600; Vector, Burlingame, CA) as secondary antibody, diaminobenzidine as chromogen, and Eukitt (O. Kindler, Freiburg, Germany) as mounting medium. X-gal- and immunostained brain sections were visualized using bright-field microscopy (Axioplan; Zeiss, Göttingen, Germany). Images were acquired with a charge-coupled device (CCD) camera (INTAS, Göttingen, Germany) and processed using Adobe Photoshop 5.0.2 (Adobe Systems, San Jose, CA) and Canvas 7.0.2 (Deneba Systems, Miami, FL) software.

Brain Slice Preparation for Electrophysiology and Secretion Experiments
For electrophysiology and secretion experiments, mice were anesthetized with halothane (Eurim-Pharm, Piding, Germany) and then decapitated. Brains were dissected in ice-cold, gassed (95% O2/5% CO2) Ringer solution (Biometra, Göttingen, Germany), containing (in mM) 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 2 CaCl2, 1 MgCl2, and 25 glucose, 316 mOsm (pH 7.4), and cut coronally or sagitally into 300 µm (electrophysiological recordings) or 600-µm slices (GnRH collection) with a vibratome (HR-2, Sigmann Elektronik, Hüffenhardt, Germany) as described (3). Slices were transferred using the back end of a Pasteur pipette to an incubation chamber with Ringer solution (equilibrated with 95% O2/5% CO2) for 30 min at 35 C and then stored at room temperature (22 C) in the same chamber until electrophysiological recording or GnRH collection.

Electrophysiological Recording
Nucleated patch recordings were used to assess AMPAR and NMDAR function because they, unlike whole-cell recordings in slice preparations, allow for fast application of glutamate (42), which is necessary to avoid receptor desensitization that would reduce the size of the already small AMPA and NMDA responses of GnRH neurons to undetectable levels. Nucleated patch recordings were made from GFP-labeled neurons in the preoptic area in coronal slices as described (3) except that the intracellular solution contained (in mM) 140 CsCl, 2 Mg-ATP, 10 EGTA, 10 HEPES, pH adjusted to 7.30 with CsOH, 286 mOsm, supplemented with 0.1 spermine. An EPC-9 amplifier and Pulse 8.53 software (HEKA, Lambrecht, Germany) were used to acquire (10 kHz), filter (Bessel, 3.33 kHz), and analyze patch clamp data, which was stored on a Power Macintosh computer. The patch clamp amplifier was also used to compensate pipette and cell capacitance. Series resistance was uncompensated but was always less than 50 M{Omega} in recordings selected for analysis. Traces were processed for presentation using Igor 3.03 (Wavemetrics, Lake Oswego, OR) and Canvas 7.0.2 software.

Fast Application of Agonists
Agonists (glutamate and NMDA) were applied to nucleated patches via a double-barrelled pipette made from theta glass tubing as described (3). Using a peristaltic pump (IPC-N8; Ismatec, Zürich, Switzerland), one barrel of the application pipette was perfused with nominally Mg2+-free control solution consisting of (in mM) 152 NaCl, 5.8 KCl, 1.9 CaCl2, 5.4 HEPES, 10 µM glycine, pH adjusted to 7.25 with NaOH, 290 mOsm, and the other barrel with control solution containing agonists.

Evaluation of Gonadal and Sperm Morphology
Ovaries and testes were removed from 4% paraformaldehyde/PBS-perfused mice, incubated for 2 h in 1% OsO4 in PBS at room temperature, dehydrated, embedded in Epon, sliced into ultrathin (1 µm thick) sections using an ultramicrotome (Reichert, Leica Microsystems, Vienna, Austria), mounted onto slides, stained with toluidine blue, visualized with bright-field microscopy, and imaged with a CCD camera.

Semen was withdrawn from mice, pipetted onto glass slides, and the spermatozoa were stained with Diff-Quick (Baxter Dade, Dudingen, Switzerland), visualized with bright-field microscopy, and imaged with a CCD camera. Sperm motility was assessed qualitatively by F. Zimmermann [Zentrales Tier Labor (ZTL), University of Heidelberg, Heidelberg, Germany].

Determination of Serum Testosterone Levels
The method for determination of serum testosterone levels was similar to that described previously (22). Trunk blood was collected from mutant mice and control littermates after halothane anesthesia and decapitation. Blood was centrifuged at 1200 x g and then the serum-containing supernatant was aliquoted and stored at –20 C for subsequent analysis. After thawing, 10 µl serum were assayed for testosterone using an ELISA kit and the provided protocol (ICN Biomedicals, Eschwege, Germany).

Collection and Measurement of GnRH Secreted from Brain Slices
The procedure for GnRH secretion experiments was adapted from published protocols (17, 18, 19, 20, 21). To sample GnRH from as many GnRH neurons as possible, the three most medial 600-µm-thick sagittal brain slices from each mouse were placed in a 0.5 ml chamber in a 35.5 C water bath and superfused (53 µl/min) with Ringer solution (equilibrated with 95% O2/5% CO2), similar to the system used to measure GnRH pulsatility in guinea pig brain slices (17). Samples (800 µl) of effluent from the chamber were collected in 1.5-ml microfuge tubes on wet ice every 15 min, the highest sampling rate with which we could detect GnRH, beginning around 1800 h, and then frozen on dry ice. The chamber and the tubing carrying the effluent were pretreated with Sigmacote (chlorinated organopolysiloxane in heptane) to prevent sticking and loss of GnRH. At the end of 4 h, slice viability was tested by depolarization for 45 min with 37.5 mM KCl Ringer solution, in which the K+ and Na+ concentrations of the regular Ringer solution were raised and lowered by 35 mM, respectively, to stimulate secretion (43). Afterward, samples were moved to a –70 C freezer until RIAs were performed.

GnRH RIA was performed as follows: each sample was divided into two 350-µl samples and pipetted into 2-ml microfuge tubes for duplicate measurements. A volume of 150 µl of PBT [0.063 M Na2HPO4, 0.013 M EDTA, 0.02% NaN3 (pH adjusted to 7.4), supplemented with 0.1% Triton X-100 and 0.2% BSA] was placed in each tube. This was followed by addition of 50 µl of GnRH antiserum R1245 (final dilution 1:300,000; gift of Dr. T. M. Nett, Colorado State University, Fort Collins, CO), incubation at 4 C for 48 h, addition of 50 µl (equivalent to 10,000 cpm or 3 pg) of 125I-labeled GnRH tracer ](3-[125I]Iodotyrosyl5) LHRH; Amersham Biosciences Europe, Freiburg, Germany] prediluted in PBT, incubation at 4 C for another 48 h, and addition of 1.5 ml ice-cold 95% ethanol. After centrifugation at 2200 x g (4,800 rpm) at 4 C for 15 min, decanting of the supernatant, and air-drying of the pellet, the pellet was dissolved in 150 µl 5% sodium dodecyl sulfate in H2O, the solution was vortexed, 1.5 ml scintillation fluid (SolvHP; Beckman Coulter, Fullerton, CA) was added, the solution was vortexed again, and radioactivity was determined in a scintillation counter (LS6500; Beckman Coulter). For each RIA, a standard curve relating 125I radioactivity to GnRH concentration was obtained using unlabeled GnRH (Bachem Biochemica, Heidelberg, Germany) ranging from 0.03–3000 pg/ml. Assay sensitivity was 0.5 pg/tube, equivalent to 1.5 pg/ml GnRH, at 95% binding. The intraassay coefficient of variation at 88% binding, or 3 pg/ml, near the mean level (3.7 pg/ml) of GnRH secretion in our experiments, was 2.3%, and the interassay coefficient of variation was 13.5%.

GnRH secretory pulses were detected by the Cluster computer algorithm (44), using a mean intraassay coefficient of variation of 4%, equal to 1.5 times the mean coefficient of variation of GnRH values obtained in mock experiments (n = 6) in which Ringer solution containing 2 pg/ml GnRH was pumped through the slice incubation chamber. Other parameters selected for the algorithm included a 1 x 1 cluster configuration and a t-statistic of 1 for the upstroke and 1 for the downstroke of each pulse to obtain a 1% false positive rate.

Measurement of Breeding Performance and Litter Size
Breeding performance of mutant mice was determined by mating with wild-type mice. Mutant mice were considered poor breeders if no offspring were produced after mating for two months with at least two different wild-type mice (either C57BL/6 or NMRI) of the opposite sex. Litter sizes corresponded to the number of pups of each mother assessed within the first week after the birth of a litter.

Behavioral Tests
Mounting and Male Aggression.
Mounting by a mutant or control male of a wild-type female was video-monitored for 24 h after placing the female into a cage occupied by a male. Aggression was assessed after placing a wild-type male intruder mouse that had previously mated with a female into a cage occupied by a mutant or control male host. The intruder and host were kept together until an aggressive behavior from one or the other was observed, or for a maximum of 10 min.

Pup Retrieval and Maternal Aggression.
Pup retrieval tests were performed by placing pups (1–4 d old) into a corner of their cage opposite their nest, then waiting 5 min for their mutant or control littermate mother to retrieve them and place them back in their nest. Maternal aggression was determined by placing an unknown wild-type male into a cage containing a mutant or control littermate mother and her offspring (1–4 d old). The male was left in the cage until an aggressive behavior from the mother was observed, or for a maximum of 10 min.

Grip Strength, Exploration, and Anxiety.
To measure grip strength, mice were allowed to grasp a handle connected to a force-measuring device and then were pulled back with their tails until they released the handle. To measure exploratory behavior, mice were placed into an open field (1 m2), and the horizontal distance traveled in 5 min was recorded. To measure anxiety, mice were placed in a dark box and given the opportunity to move to a lit box. Parameters measured were the number of exits, time latency of the first exit from the dark compartment, and the time spent in the lit compartment.

Open Field Task to Assess Male Responsiveness to Female Pheromones/Odorants.
To determine male responsiveness to female pheromones/odorants, male mice previously exposed to females for several days were placed in a video-monitored open field (0.36 m2) with different beddings in a petri dish (8.5 cm in diameter) placed in the middle of the field. The test consisted of four different trials (3 min each) for each mouse starting without bedding, followed by clean bedding, female bedding (bedding collected from a cage that had been occupied for a week by several females), and finally clean bedding again as a control. The intertrial interval was 20–40 min. The total distance traveled (in cm) and the time spent in the middle of the field (in sec) were calculated using software (VideoMot 2) provided by TSE Systems (Bad Homburg, Germany).

Statistics
Data are expressed as mean ± SEM except as indicated. Statistical comparisons were performed using the Mann-Whitney U test for two independent groups or Kruskal-Wallis one-way ANOVA for multiple independent groups (45) with the help of GB-STAT 5.06 software (Dynamic Microsystems, Silver Spring, MD). A difference between groups was significant if the probability value (P) obtained from the Mann-Whitney U test or the {chi}2 distribution associated with the Kruskal-Wallis one-way ANOVA was less than 0.05.

Reagents
Unless indicated otherwise, reagents were obtained from Sigma-Aldrich (Deisenhofen, Germany)


   ACKNOWLEDGMENTS
 
We thank Dr. P. Soriano (Fred Hutchinson Cancer Research Center, Seattle, WA) for R26R mice, Dr. T. M. Nett (Colorado State University, Fort Collins, CO) for GnRH antiserum R1245, and F. Zimmermann (University of Heidelberg, Heidelburg, Germany), A. Herold, J. Kling, and M. Lang (all from Max-Planck-Institute for Medical Research) for technical assistance.


   FOOTNOTES
 
D.R.S. was supported by a Deutsche Forschungsgemeinschaft (DFG) long-term fellowship. D.J.S. was supported by Sonderforschungsbereich Grant 488 of the DFG.

Present address for D.J.S.: Section of Endocrinology, Department of Pediatrics, University of Chicago, 5841 South Maryland Avenue, MC 5053, Chicago, Illinois 60637-1470.

First Published Online August 11, 2005

1 D.J.S. and P.H.S. are the senior authors. Back

Abbreviations: AMPA, (S)-{alpha}-Amino-3-hydroxy-5-methyl-isoxazolepropionic acid; AMPAR, AMPA receptor; CCD, charge-coupled device; GFP, green fluorescent protein; iCre, codon-improved Cre gene; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; VGluT2, vesicular glutamate transporter 2.

Received for publication July 1, 2005. Accepted for publication August 4, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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