Sexual attraction enhances glutamate transmission in mammalian anterior cingulate cortex
© Wu et al. 2009
Received: 18 April 2009
Accepted: 06 May 2009
Published: 06 May 2009
Functional human brain imaging studies have indicated the essential role of cortical regions, such as the anterior cingulate cortex (ACC), in romantic love and sex. However, the neurobiological basis of how the ACC neurons are activated and engaged in sexual attraction remains unknown. Using transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the promoter of the activity-dependent gene c-fos, we found that ACC pyramidal neurons are activated by sexual attraction. The presynaptic glutamate release to the activated neurons is increased and pharmacological inhibition of neuronal activities in the ACC reduced the interest of male mice to female mice. Our results present direct evidence of the critical role of the ACC in sexual attraction, and long-term increases in glutamate mediated excitatory transmission may contribute to sexual attraction between male and female mice.
Romantic love is a near universal notion, and is associated with a specific set of physiological, psychological and behavioral traits, most of which are also characteristic of mammalian courtship attraction . The olfactory system has been the focus of the majority of mammalian studies, as it is essential for sex-specific behaviors such as sex discrimination, sexual attraction, mating and aggression [2–6]. In humans, numerous studies have been conducted in order to determine the neural correlates associated with romantic love. Using imaging techniques such as positron emission tomography (PET) or functional magnetic resonance imaging (fMRI), higher brain structures such as primary and associative cortical regions, including piriform, orbitofrontal, temporal and cingulate cortices have been linked to sexual arousal and romantic love [7, 8]. Therefore, sexual behaviors are thought to be associated with neural networks of cortical activation.
The anterior cingulate cortex (ACC) is one of the major cortical areas involved in both negative (such as pain and fear memory) and positive (such as pleasure and sexual arousal) affective states [9–12]. For positive affective states, activation of the ACC has been correlated with romantic love, sexual arousal, as well as sexual drive [13–16]. An earlier study also reported that electrical stimulation of sites within the ACC of macaque induced penile erections , suggesting the direct role of the ACC in sexual arousal. With the use of immediate early genes like c-fos as a marker, neurons in the ACC have been shown to be activated after sexual stimulation in rodents [18, 19]. However, the neurobiological basis of how the ACC neurons are activated and engaged in sexual attraction remains poorly understood. One potential reason is that it is difficult to identify ACC neurons that are activated by sex or sexual attraction in electrophysiological experiments. In the present study, we performed whole-cell patch- clamp recording from neurons in brain slices of transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the promoter of the activity-dependent gene c-fos. Thus, we were able to record in the first time from ACC pyramidal cells that are responsive to female mice. We found that the sexual attraction between male and female mice indeed triggered long-lasting enhancement of glutamate mediated excitatory transmission within the ACC synapses.
Two chamber behavioral test for male-female attraction
The ACC is activated and required for sexual attraction
Density of Fos-immunostained (fos +), NeuN-immunostained (NeuN+) neurons and percentages of double-labeled neurons in the ACC.
Number of immunostained cells
(% of NeuN +)
(% of Fos +)
2.8 ± 2.0
32.0 ± 5.4
8.1 ± 5.4
3.8 ± 1.7
34.8 ± 4.6
11.2 ± 5.2
52.0 ± 26.9
642.0 ± 64.6
8.1 ± 4.2
135.2 ± 13.1 (**)
678.2 ± 66.9
21.4 ± 3.9 (**)
67.0 ± 34.2
1057.4 ± 99.1
6.1 ± 3.0
166.8 ± 23.0 (**)
1011.8 ± 65.5
17.2 ± 3.2 (**)
Altered synaptic transmission in the ACC neurons after sexual attraction
We then investigated excitatory synaptic transmission by recording the excitatory postsynaptic currents (EPSCs) in layer II/III pyramidal neurons by focal stimulation in deeper layer V in the presence of GABAA antagonist, picrotoxin (100 μM). Paired-pulse facilitation (PPF) ratio, a commonly used criteria for studying presynaptic release [25, 26], was examined in three types of ACC pyramidal neurons. We found that in RS cells and IB cells, there was no difference in PPF between naïve mice and mice after sexual attraction (IB: F1:44 = 0.008, P = 0.927; RS: F1:49 = 1.206, P = 0.279, Two-way ANOVA, Fig. 4B and 4C). However, a significant decrease in PPF was found in IM cells of sexually attracted mice compared to that of naïve mice (F1:89 = 9.825, P = 0.002, Fig. 4B and 4C). These results indicate that synaptic transmission was altered in the ACC neurons after sexual attraction, especially IM cells.
The alteration in PPF is input-specific
Enhanced synaptic transmission to activated neurons in the ACC after sexual attraction
We then compared PPF of GFP-positive and GFP-negative neurons (Fig. 8C and 8D). Consistently, we found significant decreases of PPF at 35 ms and 50 ms intervals in GFP-positive neurons (35 ms interval, 0.92 ± 0.08 vs. 1.40 ± 0.19; n = 8 neurons/4 mice in each group, P < 0.05, Fig. 8E and 8F). Although we did not confirm if recorded GFP-positive neurons are IM cells, our recent data found that about 50% layer II/III pyramidal cells are IM cells (unpublished data). To confirm the possibility of enhanced presynaptic glutamate release in GFP-positive neurons, we examined the miniature EPSCs (mEPSCs). We found that mEPSC frequency in GFP-positive neurons (5.8 ± 0.8 Hz, n = 8 neurons/4 mice) was larger than in GFP-negative neurons (3.0 ± 0.5 Hz, n = 8 neurons/4 mice) (P < 0.01, Fig. 8E and 8F). However, there was no significant difference in the amplitude of mEPSCs between GFP-positive and negative neurons (n = 8 neurons/4 mice, P = 0.64, Fig. 8E and 8G). These results indicate that presynaptic glutamate release was increased in the GFP-positive neurons.
In the present study, we focused on sexual attraction and the ACC, a region known to be associated in pain and pleasure . We found that the ACC is robustly activated in male mice after exposure to female mice, as demonstrated by Fos expression, and pharmacological inhibition of the ACC could reduce sexual attraction. Although the excitability of ACC pyramidal neurons was not altered, the excitatory synaptic transmission was enhanced after sexual attraction. By using transgenic fosGFP mice, we found that sexual attraction induced functional increases of presynaptic glutamate release to activated neurons in ACC slices. Our results demonstrate the critical role of the ACC in sexual attraction, and provide the first evidence for the cellular mechanism of the ACC neurons involved in the sexual attraction.
Evidence from animal studies indicates that the central supraspinal systems controlling sexual arousal are localized predominantly in the limbic system. The ACC has been implicated as an unique area for evoking affective state such as romantic love , and a PET imaging study showed that the ACC is specifically activated during the excitation phase of sexual stimuli in men . In the present study, we used Fos expression as a marker of functional activation of neurons in the ACC. We found that all layers except layer I are activated bilaterally in the ACC, suggesting that sexual attraction results in specific pattern of activation. The results are consistent with previous reports mapping the brain areas involved in sexual attraction [19, 28], and establish the critical roles of the ACC in the sexual behaviors. However, due to limitations in using Fos as a marker, future studies must be conducted to elucidate the exact inputs to and outputs from the ACC after activation by sexual attraction. The significance of the activation of the ACC is probably not on the perception of sexual stimulation, which is fulfilled by rodent olfactory systems. Rather, we propose that the activation of the ACC affects positive emotional state, leading to sexual behaviors. In favor of the role of the ACC in sexual attraction, we found that inactivation of the ACC by local infusion of muscimol reduced the interest of male mice to female mice.
How could the ACC neurons be activated by sexual attraction? Fos expression is due to the conversion of extracellular signals into early genomic activation, which indicates that a signal transduction event has taken place in those activated neurons. Therefore, we hypothesized that functional alterations occurred in the activated neurons in the ACC after sexual attraction. To test this, we used fosGFP mice, a useful tool able to link sensory stimulation in animals to functional neuroanatomy and electrophysiology. In brain slices, we could detect GFP signals and performed whole-cell patch clamp recordings in GFP-positive neurons. We found enhanced presynaptic glutamate release, as shown by decreased PPF and increased mEPSC frequency. However, postsynaptic response was unlikely to be altered, since no changes were observed in the response mediated by either AMPA or NMDA receptors. We propose that the increased presynaptic glutamate release is the main trigger for the activation of neurons in the ACC following sexual attraction. This raises two essential questions which will require further insight into the function of the ACC in sexual attraction: (1) what signal triggers the increased glutamate release? and (2), what are the functional consequences of Fos activation?
In sum, our results provide a first mouse electrophysiological model for studying the sexual attraction in forebrain areas. At a single cell level, we are able to demonstrate a long-lasting changes in glutamate mediated excitatory synaptic transmission within the ACC, a cortical region known to be critical for sexual desire, attraction, and romantic love in human imaging studies. The use of transgenic and gene knockout mice in future will provide powerful tools to explore cellular and molecular basis of key brain functions of human such as love, sex and attraction between man and woman.
Adult C57BL/6 mice were purchased from Charles River (9–17 weeks old). The transgenic fosGFP mice were obtained from laboratory of Dr. Alison Barth (Carnegie Mellon University). All mice were maintained on a 12 h light/dark cycle with food and water provided ad libitum. All protocols used were approved by The Animal Care and Use Committee at the University of Toronto.
Sexual Attraction Test
We devised a novel test to measure the degree of sexual attraction. The testing apparatus consisted of a rectangular box with a divider in the middle. The divider had numerous holes that were large enough to allow for vision and olfaction yet small enough to prevent physical interaction. For each test, mice were placed on opposing sides of the divider and were allowed to move freely within the box for 30 minutes. The more time the animal spent in the central area (area closer to the opposite sex), the greater the sexual attraction. The movement of the mice were tracked and recorded by a video camera tracking system (Ethovision, Noldus VA). Each mouse was individually placed in the rectangular box prior to testing for 10 minutes for acclimatization. None of the animals, whether male or female, were sexually experienced prior to the experiment. Although we did not monitor the female hormone cycle stage, we found similar sex attractions between male and female mice.
Whole-cell patch-clamp recordings
Coronal brain slices (300 μm) containing the ACC from six- to eight-week-old male mice were prepared using standard methods . Slices were transferred to a submerged recovery chamber with oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM): 124 NaCl, 2.5 KCl, 2 CaCl2, 2 MgSO4, 25 NaHCO3, 1 NaH2PO4, 10 glucose at room temperature for at least 1 hour.
All electrophysiological experiments were performed at room temperature. An Olympus BX51WI microscope (Tokyo, Japan) with infrared DIC optics was used for visualization of whole-cell patch clamp recording. Excitatory postsynaptic currents (EPSCs) were recorded from layer II/III neurons with an Axon 200 B amplifier (Molecular devices, CA) and the stimulations were delivered by a bipolar tungsten stimulating electrode placed in layer V of the prefrontal slices. EPSCs were induced by repetitive stimulations (duration is 200 μs, intensity is adjusted to induce EPSCs with amplitude of 50–100 pA at 0.02 Hz and neurons were voltage clamped at -70 mV. The recording pipettes (3–5 MΩ) were filled with solution containing (mM): 145 K-gluconate, 5 NaCl, 1 MgCl2, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, and 0.1 Na3-GTP (adjusted to pH 7.2 with KOH). Alexa fluor 594 (100 μM) was added in the intracellular solution to identify the expression of fosGFP as well as neuronal morphology. When current-voltage relationships were measured, K-gluconate was replaced by equomolar CsMeSO3 and 5 QX-314 chloride was added in the internal solution. Picrotoxin (100 μM) was always present to block GABAA receptor-mediated inhibitory currents and monitored throughout the synaptic currents. Access resistance was 15–30 MΩ and was monitored throughout the experiment. Data were discarded if access resistance changed more than 15% during an experiment. Statistical comparisons were performed using the Student's t-test or two way ANOVA test.
To image GFP-positive neurons, a confocal microscope (Fluoview FV 1000, Olympus, Tokyo, Japan) was used . The laser with a wavelength of 488 nm was used for GFP excitation and 633 nm was used for Alexa fluor 594.
Ten animals, divided into two groups, were anaesthetized with isoflurane and perfused with 0.1 mol/L phosphate buffered saline (PBS, pH 7.2–7.4) via the ascending aorta followed by 4% paramaformaldehyde in 0.1 mol/L PB. The brains were then removed, and postfixed in the same fixative for 4 h before cryoprotection in PBS containing 30% sucrose overnight at 4°C. Every fourth sections of 25 μm thickness, serially cut through the brain in cryostat, were collected. Sections were then used for c-Fos and/or NeuN immunoreactivity.
Sections were sequentially incubated with the following solutions: (1) a solution of 3% bovine serum albumin (BSA, Sigma, St. Louis, USA), 0.3% Triton X-100 containing rabbit antibody against c-Fos (1:6000, Calbiochem, USA), or a mixture of rabbit antibody against c-Fos (1:6000, Calbiochem, USA) and mouse monoclonal antibody directed against NeuN (1: 400, Chemicon) for 2 days at 4°C, (2) Rhodamine-conjugated goat anti-rabbit (1:200, Chemicon), or a mixture of rhodamine-conjugated goat anti-rabbit (1:200, Chemicon) and FITC-conjugated goat anti-mouse (1:200, Jackson ImmunoResearch) antibodies in PBS containing 3% BSA and 0.3% Triton X-100 for 24 h at 4°C. For 3-3'-diaminobenzidine (DAB) reaction, biotinylated goat anti-rabbit IgG (1:200; Vector) was used in the second step and these sections were further incubated with avidin-biotin-complex (elite A, B; 1:200; Vector). All sections were rinsed with PBS (3 × 10 min) after each step. The signals were visualized under epifluorescence microscope or processed with DAB as chromogen using DAB kit (Vector, Laboratories, Burlingame, CA). No staining was observed on brain sections when the primary antibody was omitted from the protocol. Images were captured with the assistance of Image-Pro Plus 5.0 software, and all the parameters used were kept consistent during capturing. After microscopic observation, sections were then counterstained by Nissl technique for cytoarchitectural examination.
Results were analyzed by t-test or two-way ANOVA followed by post-hoc Student-Newman-Keuls test to identify significant differences. All data are expressed as mean +/- SEM. In all cases, P < 0.05 was considered statistically significant.
Supported by grants from the Canadian Institutes of Health Research (CIHR66975, CIHR84256), the EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health, and the Canada Research Chair to M. Z. L.-J.W. is supported by postdoctoral fellowships from the Canadian Institutes of Health Research and Fragile X Research Foundation of Canada. We thank Dr. Alison Barth (Carnegie Mellon University) for providing transgenic fosGFP mice.
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