Original Article
Hypothalamic Sidedness in Mitochondrial Metabolism: New Perspectives
Reproductive Sciences 2014, Vol. 21(12) 1492-1498 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719114530188 rs.sagepub.com
Istvan Toth, DVM1, David Sandor Kiss, PhD1, Greta Goszleth, MSc1, Tibor Bartha, DVM, PhD1, Laszlo V. Frenyo, DVM, PhD1, Frederick Naftolin, MD, PhD2, Tamas L. Horvath, DVM, PhD3, and Attila Zsarnovszky, DVM, PhD1
Abstract Morphofunctional changes in hypothalamic neurons are highly energy dependent and rely on mitochondrial metabolism. Therefore, mitochondrial adenosine triphosphate production plays a permissive role in hypothalamic regulatory events. Here, we demonstrated that in the female rat hypothalamus, mitochondrial metabolism and tissue oxygenation show an asymmetric lateralization during the estrous cycle. This asymmetry was not detected in males. The observed sidedness suggests that estrous cycle-linked hypothalamic functions in females are based on hemispheric distinction. The novel concept of hypothalamic asymmetry necessitates the revision of hypothalamic neural circuits, synaptic reorganization, and the role of hypothalamic sides in the regulation of integrated homeostatic functions. Keywords hypothalamus, mitochondrial respiration, metabolic asymmetry, estrous cycle, rat
Introduction The brain is an organ with symmetric tissue organization. Because of its symmetrical nature, there are basically 2 types of brain structures: paired areas on the 2 sides of the brain and unpaired structures along the anatomical midline. In the mature organism, paired brain areas usually have distinct physiological functions. The first known reports on functional cerebral asymmetry were published in 1861.1 Since then, it became clear that cerebral regions are specialized to distinct functions, that is, each of the cerebral hemispheres dominate in certain specific functions. In line with these discoveries, few authors reported on the asymmetric lateralization of the rat neuroendocrine system, particularly in the hypothalamus–gonad axis.2 For example, Gerendai and Halasz described that unilateral ovariectomy resulted in asymmetric changes in hypothalamic protein 3 and gonadotrophin hormone-releasing hormone (GnRH)4 content in rats. Klein and Burden found that in a significant majority of rats, the right-sided ovary is more richly innervated by sympathetic fibers than the left.5 Supporting these findings, lesion studies suggested that an asymmetry exists in the hypothalamic control of the ovarian cycle.6-9 Glick et al provided evidence that there is more metabolic activity on the right side of the rat hypothalamus.10 Thus, although reported by only a few research groups, evidence exists for the anatomical, hormonal, and metabolic laterality in the rat hypothalamus. Unfortunately, those early findings received little attention. The history of hypothalamus-based research testifies that
the hypothalamus, a crossroad and center of many homeostatic regulatory pathways, has most frequently been investigated as an unpaired midline structure, despite its seemingly symmetric histological characteristics. Given that the cyclic activity of the female hypothalamus periodically enters the state of high energy (adenosine triphosphate [ATP]) need, we hypothesized that if functional hypothalamic asymmetry existed, at some point of the reproductive cycle, it should be detectable on the level of a general parameter of neuronal metabolism, the mitochondrial respiration. In the hypothalamus, the cycles of female reproductive functions and the alternating on–off ‘‘cycles’’ of the neural initiation of food intake are reflected by the concurrent reorganization of synapses.11-14 Synaptogenesis and neurotransmission are highly energy dependent.15 Neuronal mitochondria, as cellular energy sources, are major players in fueling neuronal functions, and the adjustment of their energy (ATP) production to the actual needs of
1 Department of Physiology and Biochemistry, Szent Istvan University Faculty of Veterinary Sciences, Budapest, Hungary 2 Reproductive Biology Research, New York University, New York, NY, USA 3 Division of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA
Corresponding Author: Attila Zsarnovszky, Department Physiology & Biochemistry, Szent Istvan University Faculty of Veterinary Sciences, Istvan u. 2., Budapest 1078, Hungary. Email: [email protected]
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hypothalamic circuits is of utmost importance. Therefore, we investigated mitochondrial respiration rates (mrrs) in left and right sides of hypothalami isolated from normal cycling female rats, under various experimental conditions (that are heretofore referred to as state 1 to 5 (St1-5) mitochondrial respiration, please see explanation subsequently) throughout the estrous cycle and compared the results to those obtained from intact male rats. Results show a clear hypothalamic asymmetry in females, but not in males, and strongly suggest that in females, functionally adjoined mechanisms (such as blood supply/tissue oxygenation) support and maintain the observed hypothalamic laterality.
Materials and Methods Intact male and normal cycling female Wistar rats were used. Following the guidelines laid down by the National Institutes of Health, the use of animals was approved by the University Committee on Animal Use at Szent Istvan University Faculty of Veterinary Sciences, Hungary.
Brain Synaptosomal/Mitochondrial Preparation and Measurement of Oxygen Consumption Reproductive cycling was determined and verified by periodic examination of vaginal smears (daily tests for 3 consecutive cycles). Collection of hypothalamic samples was started 2 weeks after the determination of cyclicity ended only using those animals whose cycling was considered physiological (cycles of 4-5 days), thus possible influential effects of vaginal smearing on cyclicity could be discounted. All (male and female) brains were removed after quick guillotine decapitation between 9:00 and 10:00 AM, and the actual estrous phase was determined based on the cytology of vaginal smears. It is noteworthy that our rationale for the postmortem vaginal smearing was to avoid hormonal influence through the possible mechanical irritation of the cervix; however, this also resulted in the difference in the number of animals in different estrous phases. Vaginal smears were evaluated on the basis of the following criteria: Early proestrus (EP): many epithelial cells þ few cornified cells (n ¼ 13). Late proestrus (LP): many epithelial cells þ many cornified cells (n ¼ 8). Estrus (E): many cornified cells with or without few epithelial cells (n ¼ 9). Metestrus (ME): many leucocytes þ few epithelial cells with or without few cornified cells; or many leucocytes þ few cornified cells with or without many epithelial cells (n ¼ 24). Diestrus (DE): few leucocytes þ few epithelial cells with or without few cornified cells (n ¼ 12). Males (n ¼ 10). Hypothalami were dissected from the removed brains as follows: in anterioposterior direction: between the caudal
margin of the optic chiasm and the rostral margin of the mamillary body and in dorsoventral direction: below the upper margin of the fornix. Dissected hypothalami were then cut into left and right halves. Hypothalamic samples were homogenized in isolation buffer (215 mmol/L mannitol, 75 mmol/L sucrose, 0.1% fatty acid-free bovine serum albumin, 20 mmol/L hydroxyethyl piperazineethanesulfonic acid [HEPES], 2 mmol/L MgCl, and 2.5 mmol/L KH2PO4, pH adjusted to 7.2 with KOH). The homogenate was spun at 1300g for 3 minutes, the supernatant was removed, and the pellet was resuspended with isolation buffer and spun again at 1300g for 3 minutes. The 2 sets of supernatants from each sample were topped off with isolation buffer and centrifuged at 13 000g for 10 minutes. The supernatant was discarded; the pellet was resuspended with isolation buffer and layered on 15% Percoll (GE Healthcare Life Sciences). The next centrifugation step was to separate the synaptosomal and perikaryal mitochondrial fractions from cell debris at 22 000g for 7 minutes. After this procedure, the final 1 mL of centrifugate contained 3 layers/fractions: the perikaryal mitochondrial fraction is at the lower tip of the tube, the middle layer is the synaptosomal fraction, and the top layer was the myelin-rich debris. Since myelin can mask the results of mitochondrial respiration, this fraction has been omitted from further sampling. Thus, the lower 200 mL of the centrifugates, containing the mitochondrial and synaptosomal fractions, were resuspended and topped off with isolation buffer without ethylene glycol tetraacetic acid (EGTA) and centrifuged again at 22 000g for 7 minutes. The supernatant was discarded; the pellet was resuspended in isolation buffer without EGTA and spun at 13 000g for 10 minutes. As the last step of the separation procedure, the supernatant was poured off, and the pellet was stored on ice till the mitochondrial oxygen-consumption measurement. Before the measurement, equal volumes (50 mL) of the samples were placed in the electrode chamber (Clark-type oxygen electrode; Hansatech Instruments, Norfolk, United Kingdom) at 37 C and diluted with 450 mL respiration buffer. Measured values represent the mrr (given in consumed nmol O2/mL of final/measured volume).
Definition of Mitochondrial Respiration States As the name and numeral marking of different mitochondrial respiration states varies in the relevant literature, subsequently we explain our nomenclature as used in the present study. Explanation of mitochondrial respiration states as sequentially measured (60 seconds for each respiration state): First step: the mitochondrial oxygen consumption was measured in respiration buffer only, without the addition of any substrates that may affect mitochondrial respiration. Under such conditions, oxygen consumption per unit time depends on the actual metabolic state of the hypothalamic sample and the sample’s original oxygen supply. We termed this experimental setup as state 1 (St1) mitochondrial respiration. Second step: to fuel the Krebs cycle, 5 mL pyruvate (P; comprised the following mixture: 275 mg pyruvate/5 mL
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distilled water þ 100 mL 1 mol/L HEPES) and 2.5 mL malate (M; comprised the following mixture: 335.25 mg malate/5 mL distilled water þ 100 mL 1 mol/L HEPES) were added to the sample. Under such conditions, the Krebs cycle intensifies and oxygen consumption increases due to consequential facilitation of the terminal oxidation and oxidative phosphorylation if the prior (in vivo) blood/oxygen supply of the hypothalamic tissue sampled was sufficient and downregulating mechanisms are not active. We termed this experimental setup as state 2 (St2) mitochondrial respiration. Third step: adenosine diphosphate (ADP; comprised the following mixture: 64.1 mg ADP/5 mL distilled water þ 100 mL 1 mol/L HEPES) of 2.5 mL was added to the sample. Since ADP is a major upregulator of mitochondrial respiration, under such conditions mrr increases if prior (in vivo) blood/fuel supply of the hypothalamic tissue was sufficient. We termed this experimental setup as (ADP dependent) state 3 (St3) mitochondrial respiration. Fourth step: oligomycin (comprised the following mixture: 1 mg oligomycin/1 mL ethanol) of 1 mL was added to the sample. Oligomycin is an ATP-synthase blocker, therefore, inhibits the oxidative phosphorylation (ATP synthesis), while terminal oxidation continues. Under such conditions, oxygen consumption depends on the actual uncoupled stage and alternative oxidation in mitochondria. Uncoupling and alternative oxidation play important roles in transient downregulation of ATP biosynthesis when cellular energy needs drop. Therefore, increased oxygen consumption in this case refers to the decline of a process (that was previously upregulated) or the attempt by the mitochondrion to downregulate ATP synthesis. We termed this experimental setup as state 4 (St4) mitochondrial respiration. Fifth step: carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP; comprised the following mixture: 1.271 mg FCCP/5 mL dimethyl sulfoxide) of 3 mL was added to the sample. The FCCP is a cyanide derivative; therefore, by binding to, and blocking cytochrome C oxidase, depletes all remaining oxygen from the sample. Decrease in oxygen level under such conditions depends on the actual/initial (in vivo) metabolic state of the sampled tissue and the amount of oxygen consumed during St1-4. Thus, the total amount of oxygen consumed in St1-4 plus the amount of remaining oxygen depleted by FCCP gives good reference to the blood/oxygen supply of the tissue at the time of the animal’s sacrifice. Therefore, this experimental setup is also known as total mitochondrial respiratory capacity, hereby referred to as state 5 (St5) mitochondrial respiration.
Data Analysis As expected, comparison of data from left and right sides of the female rat hypothalami showed that in all individuals, 1 of the 2 sides was metabolically more active so as to follow the actual estrous phase. This means that one of the hypothalamic sides of each animal displayed a nearly steady activity throughout all respiration states, with only negligible differences in mrr values (hereby termed the ‘‘silent’’ side). 1494
Figure 1. Hypothalamic asymmetry in state 1 to 5 (St1-5) mitochondrial respiration throughout the estrous cycle. Results clearly show that hypothalamic metabolic sidedness can be observed during the estrous cycle; however, the degree of laterality highly depends on the actual phase of the estrous.
Mitochondrial respiration rates measured in the contralateral (‘‘active’’) side highly varied compared to values from the contralateral side, depending on the estrous phase and the respiration state. Sidedness was considered if 60% or more of the O2 was consumed by either of the sides. Whether sidedness was statistically significant was determined by Fisher exact test (please see Figure 1), while the proportion of either left or right sidedness within the cohort of sided animals was expressed in percentage.
Results and Discussion Hypothalamic Asymmetry in Mitochondrial Metabolism in Female Rats In general, there are 2 important aspects of our results: (1) the mitochondrial metabolism showed a fluctuation that corresponded with the phases of the estrous cycle and (2) the fluctuation in mitochondrial metabolism, depending on the estrous phase and respiration state, occurred with 1-sided dominance (referred to as the ‘‘active’’ side), as shown and discussed subsequently. Therefore, it is reasonable to assume that the regulation of GnRH secretion/release is based on asymmetric (sided) hypothalamic activity. We are aware that presently there is no direct evidence available to prove whether functional inhibition of the ‘‘active side’’ would prevent the GnRH surge (ongoing experiments in our laboratory aim to clarify this question). However, for the sake of creating a new hypothesis/theory from the present results, we will attempt to interpret the data assuming that the plastic mitochondrial metabolic activity of the ‘‘active’’ side versus the
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‘‘silent’’ side is responsible for the generation of GnRH release.
The Extent of Hypothalamic Asymmetry Analysis of mitochondrial metabolism in EP animals (Figure 1) revealed that about half of the animals showed hypothalamic asymmetry (with either left- or right-sided dominance) in basic (St1) and fuel-dependent (St2) respiration states. However, the ADP-dependent St3 and uncoupled respirations (St4) were more intense in either one of the hemispheres. The highest degree of asymmetry was found in total mitochondrial respiratory capacity (St5). It should be noted that addition of pyruvate and malate (‘‘fuel,’’ P þ M) could not increase the mrr in St2 (please compare to St1 vs St2 in LP). In contrast, addition of ADP increased St3 mrr in one of the hypothalamic sides, suggesting that a unilateral mechanism exists that facilitates mitochondrial metabolism in EP, and that this mechanism is ADP dependent. Such a mechanism seems to be absent in the contralateral (‘‘silent’’) hemisphere. These results, with special regard to St5 values, suggest that there is a high degree of asymmetry in hypothalamic blood/O2 supply during EP, which may be consonant with earlier findings that the sympathetic nervous system shows asymmetry in the ovaries5 and thus, this may be a possibility in the hypothalamus as well. The degree and pattern of sidedness over St1-5 were remarkably different in LP than in EP (Figure 1). The St1 increased, compared to the EP value, and St2 was even higher (than in EP). It is important to keep in mind that experimental fueling of the mitochondria (addition of P þ M) in St2 has been done in all estrus phases, yet, in EP St2, laterality was not higher than in St1. This suggests that a yet unknown mechanism exists that can activate fuel-dependent mitochondrial respiration. It is also important that, although basic respiration increased from EP to LP, experimental fuel addition induced an even more intense respiration, suggesting that in LP, the mitochondria ‘‘are able’’ to reach more intense levels of metabolism in the potential case of need. In contrast, laterality in ADP-dependent St3 substantially decreased (in more than 30% of animals that showed St3 asymmetry) in EP. To give a suitable interpretation for this observation, one should consider the high St4 values (please see earlier explanation) and the simultaneous synaptic events that occur during the estrous cycle.11 In estrus (E), base mitochondrial metabolism (St1) was highly asymmetric, indicating that one of the sides was metabolically more active than the contralateral side in almost 90% of animals. This asymmetry decreased substantially after administration of P þ M (St2) and even more so after ADP addition (St3), suggesting that a downregulating mechanism, supposedly reflected in high LP St4 values, could bring E-related neural actions to a halt. In ME, sidedness in base metabolism (St1) decreased and a negative feedback-like regulation was still observed in St2-3, as mrr values decreased after addition of P þ M (St2) and ADP (St3). Higher than E, St4 values may indicate
Figure 2. Left–right share in hypothalamic asymmetry in early proestrus (EP). Although basic mitochondrial respiration was only sided in half of the animals (St1), addition of pyruvate and malate (St2) and adenosine diphosphate (ADP; St3) increased the proportion of sided animals to 66.66% and 77.78%. St1 indicates state 1; St2, state 2; St3, state 3.
intensifying downregulation in the ‘‘active’’ side. This idea is supported by further decreasing base mrr in DE St1. In DE, the downregulating effects of P þ M (St2) and ADP (St3) are still detectable, albeit at a smaller extent than in ME. The St4 values are also substantially decreased compared to E, likely playing a role in turning the negative feedbacklike effect of ADP to a positive, facilitatory effect (in sidedness) by the onset of EP.
The Share of the Left and Right Sides in Hypothalamic Sidedness In EP animals (Figure 2), left sidedness and right sidedness was balanced in basic mitochondrial metabolism (St1). However, when adding P þ M (St2), a right-sided dominance evolved with an increase in the right side and a decrease in the left side. This observation suggests that a mechanism exists that may inhibit metabolism on the left side but facilitate it on the right side (interactive regulation between the hemispheres). A further increase in the degree of right sidedness was found after the administration of ADP (St3), which could arise from the facilitatory effect of ADP on mitochondrial metabolism. Such an ADP effect may have worked in St2 as well, and/or in St3, we observed the additive effect of ADP and a supposed mechanism mentioned earlier regarding our St2 finding. Right sidedness in St4, based on the interpretation of the meaning of St1-5 (please see Materials and Methods section), indicates that a more intense downregulation is in progress on the right side. In spite of increased right-sided O2 consumption, St5 values show that in EP, there is still more O2 left in the right side than in the left, suggesting that the right side is metabolically more prepared for the LP. In LP (Figure 3), right sidedness was observed in basic mitochondrial metabolism (St1). This phenomenon is likely the
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Figure 3. Left–right share in hypothalamic asymmetry in late proestrus (LP). Basic mitochondrial metabolism (St1) was sided in 60% of animals. Addition of pyruvate and malate (St2) increased the proportion of sided animals to 85.7%, and adenosine diphosphate (ADP) administration (St3) resulted in full sidedness of the animals. Note that with respect to St4, full sidedness was observed, indicating that downregulating mechanisms are activated in the ‘‘active’’ hypothalamic side of all animals. In spite of the robust sidedness, only 50% of animals were sided regarding their residual hypothalamic oxygen content. St1 indicates state 1; St2, state 2; St3, state 3; St4; state 4.
Figure 4. Left–right share in hypothalamic asymmetry in estrus (E). The potentials for full right sidedness in LP St2-3 appear to be realized in E St1, where full right sidedness was detected. Interestingly, addition of P þ M in St2 resulted in a robust decrease in right sidedness accompanied by elevated left-sided metabolism. Addition of ADP in St3 reinstated the full right sidedness, supporting the idea (mentioned in LP St4 and E St2 discussion) that ADP may be the major regulator of metabolism, especially in the ‘‘active’’ side. In St4, we found a leftsided dominance. In E, less residual O2 remained in the right side (St5) versus the left side. ADP indicates adenosine diphosphate; LP, late proestrus; St1, state 1; St2, state 2; St3, state 3; St4; state 4.
consequence of the right-sided facilitatory effect of ADP having its onset in EP. Fuel-dependent asymmetry in St2 supports this idea and indicates the high potential of the right side to further increase the intensity of metabolism. Addition of ADP (St3) resulted in full right sidedness, further suggesting the facilitatory role of ADP in mitochondrial metabolism and the likely 1496
interaction between the hemispheres in a ‘‘contralateral inhibition’’ fashion. Simultaneously, full right sidedness was detected in St4, meaning that sided mitochondrial metabolism seen in St1 is accompanied with the activation of downregulating mechanisms. Based on this observation, one may anticipate that in successive estrous phases, stimulatory metabolic effects will fade. Total sidedness in St4 rises the possibility that the ‘‘passivity’’ of the silent hemisphere is the result of the lack of stimulation rather than that of some sort of inhibition. It is interesting to note that after exhausting the samples through St1-4, St5 values are fairly balanced. This means that the surplus in right-sided blood/O2 supply is proportional to the excess metabolic potentials of the right side over the left side. The potentials for full right sidedness in LP St2-3 appear to be realized in E St1, where full right sidedness was detected (Figure 4). Interestingly, addition of P þ M in St2 resulted in a robust decrease in right sidedness accompanied by elevated left-sided metabolism. This phenomenon supports our notion regarding LP St4 and may result from decreased amounts of hypothalamic ADP (and consequential weakened facilitation) on the right side. Addition of ADP in St3 reinstated the full right sidedness, supporting the idea (mentioned in LP St4 and E St2 discussion) that ADP may be the major regulator of metabolism, especially in the ‘‘active’’ side. In St4, we found a left-sided dominance. The stronger downregulation in the left side may be responsible for the maintenance of the more intense mitochondrial metabolism in the right side, and at the same time this rises the possibility that a leftsided (ie, contralateral) inhibitory mechanism may exist with a yet unidentified nature. In E, less residual O2 remained in the right side (St5) versus the left side. One may speculate that a rightsided decrease in blood/O2 supply might occur in E and that this decrease may set a limit to the fuel/O2 consumption observed in the right side, thereby bringing E to a halt. Indeed, in ME (Figure 5), the base mitochondrial metabolism (St1) seems to be balanced between the 2 hemispheres, nevertheless, addition of ‘‘fuel’’ or ADP (St2-3) shows that the right side continues to possess the potentials for a more intense metabolism compared to the left. Also, the aforementioned potential left-sided inhibition (in E St2) does not seem to act in ME. The St4 results indicate that in ME, balanced base metabolism (St1) is also accompanied by balanced downregulatory processes (St4) in the 2 hemispheres. The St5 values suggest that blood/O2 supply in the hypothalamic sides has not changed compared to E. In DE (Figure 6), right-sided dominance was apparent (St1), which could not be further increased by the addition of P þ M and/or ADP (St2-3). We currently do not know the explanation of this phenomenon; however, increased downregulation (St4) in the right side was also observed that, with lower residual O2 left in the right side (St5), may play a role in equalizing the left–right balance by the onset of EP. Mitochondrial respiration measurements in hypothalamic sides of intact males showed nearly equal (no significant differences found) mrr values for both sides (data not shown). Thus, results indicate that hypothalamic asymmetry, at least regarding mitochondrial metabolism, exists only in females, but not in males.
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Figure 5. Left–right share in hypothalamic asymmetry in metestrus (ME). Base mitochondrial metabolism (St1) seems to be balanced between the 2 hemispheres, nevertheless, addition of ‘‘fuel’’ or adenosine diphosphate (ADP; St2-3) shows that the right side continues to possess the potentials for a more intense metabolism compared to the left. Also, the potential left-sided inhibition (in E St2) does not seem to act in ME. The St4 results indicate that in ME, balanced base metabolism (St1) is also accompanied by balanced downregulatory processes (St4) in the 2 hemispheres. The St5 values suggest that blood/O2 supply in the hypothalamic sides has not changed compared to E. St1 indicates state 1; St2, state 2; St3, state 3; St4; state 4.
Figure 6. Left–right share in hypothalamic asymmetry in diestrus (DE). Right-sided dominance was apparent (St1), which could not be further increased by the addition of P þ M and/or adenosine diphosphate (ADP; St2-3). We currently do not know the explanation of this phenomenon; however, increased downregulation (St4) in the right side was also observed that, with lower residual O2 left in the right side (St5), may play a role in equalizing the left–right balance by the onset of EP. St1 indicates state 1; St2, state 2; St3, state 3; St4; state 4.
Conclusion Histologically, the hypothalamus comprises like-named nuclei with symmetrical localization on the 2 sides of the third ventricle. Although early studies indicated the possibility of functional lateralization of the hypothalamus, those findings have been ignored by successive hypothalamic research that
Figure 7. Schematic of the possible structural basis of the hypothalamic metabolic lateralization. In female rats, an estrus phase-dependent and dynamically changing metabolic asymmetry was observed with either right- or left-sided dominance during the estrous cycle and in different experimental conditions. These dynamic changes suggest that certain mechanisms may exist that enable the dominant functions of one of the sides with the simultaneous inhibition of the contralateral side. Such a ‘‘give-or-take’’ mechanism assumes that there is some kind of interconnection between the hypothalamic hemispheres through which the stimulation of one side is functionally synchronized with the simultaneous inhibition of the contralateral side. The scheme summarizes the potential information channels between the 2 hypothalamic hemispheres through which the functional synchronization of the sided functions could be controlled. These include one or more of the following: (1) anatomical interconnection through the interthalamic adhesion and/or (2) the infundibular region; and (3) humoral interconnection through the lateral evaginations of the third ventricle; possibly an asymmetric sympathetic inhibition. We termed the hypothesized synchronizing mechanism ‘‘temporal, functional bilateral synchronization.’’
continued to consider the hypothalamus as an unpaired midline structure. Our present results, however, indicate that there is a sidedness in female, but not in male, hypothalamic functions, and because of its dependence on the estrous phase, it is most likely related to the regulation of GnRH secretion/release. These observations also raise the possibility that future consideration of hypothalamic functional laterality in hypothalamus research could lead to radically new observations regarding probably all known hypothalamic functions. A hypothesized structural basis of the mechanism that enables the stimulation of a hypothalamic side and the simultaneous contralateral inhibition is shown in Figure 7.
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Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: OTKA (Hungarian Scientific Research Fund) 81745 to Attila Zsarnovszky, OTKA 72186 to Tibor Bartha, OTKA 104982 to Tibor Bartha, and TAMOP (Social Renewal Operational Programme) 4.2.2. b-10/12010-0011 to David Sandor Kiss.
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Hypothalamic Sidedness in Mitochondrial Metabolism: New Perspectives
Reproductive Sciences 2014, Vol. 21(12) 1492-1498 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719114530188 rs.sagepub.com
Istvan Toth, DVM1, David Sandor Kiss, PhD1, Greta Goszleth, MSc1, Tibor Bartha, DVM, PhD1, Laszlo V. Frenyo, DVM, PhD1, Frederick Naftolin, MD, PhD2, Tamas L. Horvath, DVM, PhD3, and Attila Zsarnovszky, DVM, PhD1
Abstract Morphofunctional changes in hypothalamic neurons are highly energy dependent and rely on mitochondrial metabolism. Therefore, mitochondrial adenosine triphosphate production plays a permissive role in hypothalamic regulatory events. Here, we demonstrated that in the female rat hypothalamus, mitochondrial metabolism and tissue oxygenation show an asymmetric lateralization during the estrous cycle. This asymmetry was not detected in males. The observed sidedness suggests that estrous cycle-linked hypothalamic functions in females are based on hemispheric distinction. The novel concept of hypothalamic asymmetry necessitates the revision of hypothalamic neural circuits, synaptic reorganization, and the role of hypothalamic sides in the regulation of integrated homeostatic functions. Keywords hypothalamus, mitochondrial respiration, metabolic asymmetry, estrous cycle, rat
Introduction The brain is an organ with symmetric tissue organization. Because of its symmetrical nature, there are basically 2 types of brain structures: paired areas on the 2 sides of the brain and unpaired structures along the anatomical midline. In the mature organism, paired brain areas usually have distinct physiological functions. The first known reports on functional cerebral asymmetry were published in 1861.1 Since then, it became clear that cerebral regions are specialized to distinct functions, that is, each of the cerebral hemispheres dominate in certain specific functions. In line with these discoveries, few authors reported on the asymmetric lateralization of the rat neuroendocrine system, particularly in the hypothalamus–gonad axis.2 For example, Gerendai and Halasz described that unilateral ovariectomy resulted in asymmetric changes in hypothalamic protein 3 and gonadotrophin hormone-releasing hormone (GnRH)4 content in rats. Klein and Burden found that in a significant majority of rats, the right-sided ovary is more richly innervated by sympathetic fibers than the left.5 Supporting these findings, lesion studies suggested that an asymmetry exists in the hypothalamic control of the ovarian cycle.6-9 Glick et al provided evidence that there is more metabolic activity on the right side of the rat hypothalamus.10 Thus, although reported by only a few research groups, evidence exists for the anatomical, hormonal, and metabolic laterality in the rat hypothalamus. Unfortunately, those early findings received little attention. The history of hypothalamus-based research testifies that
the hypothalamus, a crossroad and center of many homeostatic regulatory pathways, has most frequently been investigated as an unpaired midline structure, despite its seemingly symmetric histological characteristics. Given that the cyclic activity of the female hypothalamus periodically enters the state of high energy (adenosine triphosphate [ATP]) need, we hypothesized that if functional hypothalamic asymmetry existed, at some point of the reproductive cycle, it should be detectable on the level of a general parameter of neuronal metabolism, the mitochondrial respiration. In the hypothalamus, the cycles of female reproductive functions and the alternating on–off ‘‘cycles’’ of the neural initiation of food intake are reflected by the concurrent reorganization of synapses.11-14 Synaptogenesis and neurotransmission are highly energy dependent.15 Neuronal mitochondria, as cellular energy sources, are major players in fueling neuronal functions, and the adjustment of their energy (ATP) production to the actual needs of
1 Department of Physiology and Biochemistry, Szent Istvan University Faculty of Veterinary Sciences, Budapest, Hungary 2 Reproductive Biology Research, New York University, New York, NY, USA 3 Division of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA
Corresponding Author: Attila Zsarnovszky, Department Physiology & Biochemistry, Szent Istvan University Faculty of Veterinary Sciences, Istvan u. 2., Budapest 1078, Hungary. Email: [email protected]
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hypothalamic circuits is of utmost importance. Therefore, we investigated mitochondrial respiration rates (mrrs) in left and right sides of hypothalami isolated from normal cycling female rats, under various experimental conditions (that are heretofore referred to as state 1 to 5 (St1-5) mitochondrial respiration, please see explanation subsequently) throughout the estrous cycle and compared the results to those obtained from intact male rats. Results show a clear hypothalamic asymmetry in females, but not in males, and strongly suggest that in females, functionally adjoined mechanisms (such as blood supply/tissue oxygenation) support and maintain the observed hypothalamic laterality.
Materials and Methods Intact male and normal cycling female Wistar rats were used. Following the guidelines laid down by the National Institutes of Health, the use of animals was approved by the University Committee on Animal Use at Szent Istvan University Faculty of Veterinary Sciences, Hungary.
Brain Synaptosomal/Mitochondrial Preparation and Measurement of Oxygen Consumption Reproductive cycling was determined and verified by periodic examination of vaginal smears (daily tests for 3 consecutive cycles). Collection of hypothalamic samples was started 2 weeks after the determination of cyclicity ended only using those animals whose cycling was considered physiological (cycles of 4-5 days), thus possible influential effects of vaginal smearing on cyclicity could be discounted. All (male and female) brains were removed after quick guillotine decapitation between 9:00 and 10:00 AM, and the actual estrous phase was determined based on the cytology of vaginal smears. It is noteworthy that our rationale for the postmortem vaginal smearing was to avoid hormonal influence through the possible mechanical irritation of the cervix; however, this also resulted in the difference in the number of animals in different estrous phases. Vaginal smears were evaluated on the basis of the following criteria: Early proestrus (EP): many epithelial cells þ few cornified cells (n ¼ 13). Late proestrus (LP): many epithelial cells þ many cornified cells (n ¼ 8). Estrus (E): many cornified cells with or without few epithelial cells (n ¼ 9). Metestrus (ME): many leucocytes þ few epithelial cells with or without few cornified cells; or many leucocytes þ few cornified cells with or without many epithelial cells (n ¼ 24). Diestrus (DE): few leucocytes þ few epithelial cells with or without few cornified cells (n ¼ 12). Males (n ¼ 10). Hypothalami were dissected from the removed brains as follows: in anterioposterior direction: between the caudal
margin of the optic chiasm and the rostral margin of the mamillary body and in dorsoventral direction: below the upper margin of the fornix. Dissected hypothalami were then cut into left and right halves. Hypothalamic samples were homogenized in isolation buffer (215 mmol/L mannitol, 75 mmol/L sucrose, 0.1% fatty acid-free bovine serum albumin, 20 mmol/L hydroxyethyl piperazineethanesulfonic acid [HEPES], 2 mmol/L MgCl, and 2.5 mmol/L KH2PO4, pH adjusted to 7.2 with KOH). The homogenate was spun at 1300g for 3 minutes, the supernatant was removed, and the pellet was resuspended with isolation buffer and spun again at 1300g for 3 minutes. The 2 sets of supernatants from each sample were topped off with isolation buffer and centrifuged at 13 000g for 10 minutes. The supernatant was discarded; the pellet was resuspended with isolation buffer and layered on 15% Percoll (GE Healthcare Life Sciences). The next centrifugation step was to separate the synaptosomal and perikaryal mitochondrial fractions from cell debris at 22 000g for 7 minutes. After this procedure, the final 1 mL of centrifugate contained 3 layers/fractions: the perikaryal mitochondrial fraction is at the lower tip of the tube, the middle layer is the synaptosomal fraction, and the top layer was the myelin-rich debris. Since myelin can mask the results of mitochondrial respiration, this fraction has been omitted from further sampling. Thus, the lower 200 mL of the centrifugates, containing the mitochondrial and synaptosomal fractions, were resuspended and topped off with isolation buffer without ethylene glycol tetraacetic acid (EGTA) and centrifuged again at 22 000g for 7 minutes. The supernatant was discarded; the pellet was resuspended in isolation buffer without EGTA and spun at 13 000g for 10 minutes. As the last step of the separation procedure, the supernatant was poured off, and the pellet was stored on ice till the mitochondrial oxygen-consumption measurement. Before the measurement, equal volumes (50 mL) of the samples were placed in the electrode chamber (Clark-type oxygen electrode; Hansatech Instruments, Norfolk, United Kingdom) at 37 C and diluted with 450 mL respiration buffer. Measured values represent the mrr (given in consumed nmol O2/mL of final/measured volume).
Definition of Mitochondrial Respiration States As the name and numeral marking of different mitochondrial respiration states varies in the relevant literature, subsequently we explain our nomenclature as used in the present study. Explanation of mitochondrial respiration states as sequentially measured (60 seconds for each respiration state): First step: the mitochondrial oxygen consumption was measured in respiration buffer only, without the addition of any substrates that may affect mitochondrial respiration. Under such conditions, oxygen consumption per unit time depends on the actual metabolic state of the hypothalamic sample and the sample’s original oxygen supply. We termed this experimental setup as state 1 (St1) mitochondrial respiration. Second step: to fuel the Krebs cycle, 5 mL pyruvate (P; comprised the following mixture: 275 mg pyruvate/5 mL
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distilled water þ 100 mL 1 mol/L HEPES) and 2.5 mL malate (M; comprised the following mixture: 335.25 mg malate/5 mL distilled water þ 100 mL 1 mol/L HEPES) were added to the sample. Under such conditions, the Krebs cycle intensifies and oxygen consumption increases due to consequential facilitation of the terminal oxidation and oxidative phosphorylation if the prior (in vivo) blood/oxygen supply of the hypothalamic tissue sampled was sufficient and downregulating mechanisms are not active. We termed this experimental setup as state 2 (St2) mitochondrial respiration. Third step: adenosine diphosphate (ADP; comprised the following mixture: 64.1 mg ADP/5 mL distilled water þ 100 mL 1 mol/L HEPES) of 2.5 mL was added to the sample. Since ADP is a major upregulator of mitochondrial respiration, under such conditions mrr increases if prior (in vivo) blood/fuel supply of the hypothalamic tissue was sufficient. We termed this experimental setup as (ADP dependent) state 3 (St3) mitochondrial respiration. Fourth step: oligomycin (comprised the following mixture: 1 mg oligomycin/1 mL ethanol) of 1 mL was added to the sample. Oligomycin is an ATP-synthase blocker, therefore, inhibits the oxidative phosphorylation (ATP synthesis), while terminal oxidation continues. Under such conditions, oxygen consumption depends on the actual uncoupled stage and alternative oxidation in mitochondria. Uncoupling and alternative oxidation play important roles in transient downregulation of ATP biosynthesis when cellular energy needs drop. Therefore, increased oxygen consumption in this case refers to the decline of a process (that was previously upregulated) or the attempt by the mitochondrion to downregulate ATP synthesis. We termed this experimental setup as state 4 (St4) mitochondrial respiration. Fifth step: carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP; comprised the following mixture: 1.271 mg FCCP/5 mL dimethyl sulfoxide) of 3 mL was added to the sample. The FCCP is a cyanide derivative; therefore, by binding to, and blocking cytochrome C oxidase, depletes all remaining oxygen from the sample. Decrease in oxygen level under such conditions depends on the actual/initial (in vivo) metabolic state of the sampled tissue and the amount of oxygen consumed during St1-4. Thus, the total amount of oxygen consumed in St1-4 plus the amount of remaining oxygen depleted by FCCP gives good reference to the blood/oxygen supply of the tissue at the time of the animal’s sacrifice. Therefore, this experimental setup is also known as total mitochondrial respiratory capacity, hereby referred to as state 5 (St5) mitochondrial respiration.
Data Analysis As expected, comparison of data from left and right sides of the female rat hypothalami showed that in all individuals, 1 of the 2 sides was metabolically more active so as to follow the actual estrous phase. This means that one of the hypothalamic sides of each animal displayed a nearly steady activity throughout all respiration states, with only negligible differences in mrr values (hereby termed the ‘‘silent’’ side). 1494
Figure 1. Hypothalamic asymmetry in state 1 to 5 (St1-5) mitochondrial respiration throughout the estrous cycle. Results clearly show that hypothalamic metabolic sidedness can be observed during the estrous cycle; however, the degree of laterality highly depends on the actual phase of the estrous.
Mitochondrial respiration rates measured in the contralateral (‘‘active’’) side highly varied compared to values from the contralateral side, depending on the estrous phase and the respiration state. Sidedness was considered if 60% or more of the O2 was consumed by either of the sides. Whether sidedness was statistically significant was determined by Fisher exact test (please see Figure 1), while the proportion of either left or right sidedness within the cohort of sided animals was expressed in percentage.
Results and Discussion Hypothalamic Asymmetry in Mitochondrial Metabolism in Female Rats In general, there are 2 important aspects of our results: (1) the mitochondrial metabolism showed a fluctuation that corresponded with the phases of the estrous cycle and (2) the fluctuation in mitochondrial metabolism, depending on the estrous phase and respiration state, occurred with 1-sided dominance (referred to as the ‘‘active’’ side), as shown and discussed subsequently. Therefore, it is reasonable to assume that the regulation of GnRH secretion/release is based on asymmetric (sided) hypothalamic activity. We are aware that presently there is no direct evidence available to prove whether functional inhibition of the ‘‘active side’’ would prevent the GnRH surge (ongoing experiments in our laboratory aim to clarify this question). However, for the sake of creating a new hypothesis/theory from the present results, we will attempt to interpret the data assuming that the plastic mitochondrial metabolic activity of the ‘‘active’’ side versus the
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‘‘silent’’ side is responsible for the generation of GnRH release.
The Extent of Hypothalamic Asymmetry Analysis of mitochondrial metabolism in EP animals (Figure 1) revealed that about half of the animals showed hypothalamic asymmetry (with either left- or right-sided dominance) in basic (St1) and fuel-dependent (St2) respiration states. However, the ADP-dependent St3 and uncoupled respirations (St4) were more intense in either one of the hemispheres. The highest degree of asymmetry was found in total mitochondrial respiratory capacity (St5). It should be noted that addition of pyruvate and malate (‘‘fuel,’’ P þ M) could not increase the mrr in St2 (please compare to St1 vs St2 in LP). In contrast, addition of ADP increased St3 mrr in one of the hypothalamic sides, suggesting that a unilateral mechanism exists that facilitates mitochondrial metabolism in EP, and that this mechanism is ADP dependent. Such a mechanism seems to be absent in the contralateral (‘‘silent’’) hemisphere. These results, with special regard to St5 values, suggest that there is a high degree of asymmetry in hypothalamic blood/O2 supply during EP, which may be consonant with earlier findings that the sympathetic nervous system shows asymmetry in the ovaries5 and thus, this may be a possibility in the hypothalamus as well. The degree and pattern of sidedness over St1-5 were remarkably different in LP than in EP (Figure 1). The St1 increased, compared to the EP value, and St2 was even higher (than in EP). It is important to keep in mind that experimental fueling of the mitochondria (addition of P þ M) in St2 has been done in all estrus phases, yet, in EP St2, laterality was not higher than in St1. This suggests that a yet unknown mechanism exists that can activate fuel-dependent mitochondrial respiration. It is also important that, although basic respiration increased from EP to LP, experimental fuel addition induced an even more intense respiration, suggesting that in LP, the mitochondria ‘‘are able’’ to reach more intense levels of metabolism in the potential case of need. In contrast, laterality in ADP-dependent St3 substantially decreased (in more than 30% of animals that showed St3 asymmetry) in EP. To give a suitable interpretation for this observation, one should consider the high St4 values (please see earlier explanation) and the simultaneous synaptic events that occur during the estrous cycle.11 In estrus (E), base mitochondrial metabolism (St1) was highly asymmetric, indicating that one of the sides was metabolically more active than the contralateral side in almost 90% of animals. This asymmetry decreased substantially after administration of P þ M (St2) and even more so after ADP addition (St3), suggesting that a downregulating mechanism, supposedly reflected in high LP St4 values, could bring E-related neural actions to a halt. In ME, sidedness in base metabolism (St1) decreased and a negative feedback-like regulation was still observed in St2-3, as mrr values decreased after addition of P þ M (St2) and ADP (St3). Higher than E, St4 values may indicate
Figure 2. Left–right share in hypothalamic asymmetry in early proestrus (EP). Although basic mitochondrial respiration was only sided in half of the animals (St1), addition of pyruvate and malate (St2) and adenosine diphosphate (ADP; St3) increased the proportion of sided animals to 66.66% and 77.78%. St1 indicates state 1; St2, state 2; St3, state 3.
intensifying downregulation in the ‘‘active’’ side. This idea is supported by further decreasing base mrr in DE St1. In DE, the downregulating effects of P þ M (St2) and ADP (St3) are still detectable, albeit at a smaller extent than in ME. The St4 values are also substantially decreased compared to E, likely playing a role in turning the negative feedbacklike effect of ADP to a positive, facilitatory effect (in sidedness) by the onset of EP.
The Share of the Left and Right Sides in Hypothalamic Sidedness In EP animals (Figure 2), left sidedness and right sidedness was balanced in basic mitochondrial metabolism (St1). However, when adding P þ M (St2), a right-sided dominance evolved with an increase in the right side and a decrease in the left side. This observation suggests that a mechanism exists that may inhibit metabolism on the left side but facilitate it on the right side (interactive regulation between the hemispheres). A further increase in the degree of right sidedness was found after the administration of ADP (St3), which could arise from the facilitatory effect of ADP on mitochondrial metabolism. Such an ADP effect may have worked in St2 as well, and/or in St3, we observed the additive effect of ADP and a supposed mechanism mentioned earlier regarding our St2 finding. Right sidedness in St4, based on the interpretation of the meaning of St1-5 (please see Materials and Methods section), indicates that a more intense downregulation is in progress on the right side. In spite of increased right-sided O2 consumption, St5 values show that in EP, there is still more O2 left in the right side than in the left, suggesting that the right side is metabolically more prepared for the LP. In LP (Figure 3), right sidedness was observed in basic mitochondrial metabolism (St1). This phenomenon is likely the
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Figure 3. Left–right share in hypothalamic asymmetry in late proestrus (LP). Basic mitochondrial metabolism (St1) was sided in 60% of animals. Addition of pyruvate and malate (St2) increased the proportion of sided animals to 85.7%, and adenosine diphosphate (ADP) administration (St3) resulted in full sidedness of the animals. Note that with respect to St4, full sidedness was observed, indicating that downregulating mechanisms are activated in the ‘‘active’’ hypothalamic side of all animals. In spite of the robust sidedness, only 50% of animals were sided regarding their residual hypothalamic oxygen content. St1 indicates state 1; St2, state 2; St3, state 3; St4; state 4.
Figure 4. Left–right share in hypothalamic asymmetry in estrus (E). The potentials for full right sidedness in LP St2-3 appear to be realized in E St1, where full right sidedness was detected. Interestingly, addition of P þ M in St2 resulted in a robust decrease in right sidedness accompanied by elevated left-sided metabolism. Addition of ADP in St3 reinstated the full right sidedness, supporting the idea (mentioned in LP St4 and E St2 discussion) that ADP may be the major regulator of metabolism, especially in the ‘‘active’’ side. In St4, we found a leftsided dominance. In E, less residual O2 remained in the right side (St5) versus the left side. ADP indicates adenosine diphosphate; LP, late proestrus; St1, state 1; St2, state 2; St3, state 3; St4; state 4.
consequence of the right-sided facilitatory effect of ADP having its onset in EP. Fuel-dependent asymmetry in St2 supports this idea and indicates the high potential of the right side to further increase the intensity of metabolism. Addition of ADP (St3) resulted in full right sidedness, further suggesting the facilitatory role of ADP in mitochondrial metabolism and the likely 1496
interaction between the hemispheres in a ‘‘contralateral inhibition’’ fashion. Simultaneously, full right sidedness was detected in St4, meaning that sided mitochondrial metabolism seen in St1 is accompanied with the activation of downregulating mechanisms. Based on this observation, one may anticipate that in successive estrous phases, stimulatory metabolic effects will fade. Total sidedness in St4 rises the possibility that the ‘‘passivity’’ of the silent hemisphere is the result of the lack of stimulation rather than that of some sort of inhibition. It is interesting to note that after exhausting the samples through St1-4, St5 values are fairly balanced. This means that the surplus in right-sided blood/O2 supply is proportional to the excess metabolic potentials of the right side over the left side. The potentials for full right sidedness in LP St2-3 appear to be realized in E St1, where full right sidedness was detected (Figure 4). Interestingly, addition of P þ M in St2 resulted in a robust decrease in right sidedness accompanied by elevated left-sided metabolism. This phenomenon supports our notion regarding LP St4 and may result from decreased amounts of hypothalamic ADP (and consequential weakened facilitation) on the right side. Addition of ADP in St3 reinstated the full right sidedness, supporting the idea (mentioned in LP St4 and E St2 discussion) that ADP may be the major regulator of metabolism, especially in the ‘‘active’’ side. In St4, we found a left-sided dominance. The stronger downregulation in the left side may be responsible for the maintenance of the more intense mitochondrial metabolism in the right side, and at the same time this rises the possibility that a leftsided (ie, contralateral) inhibitory mechanism may exist with a yet unidentified nature. In E, less residual O2 remained in the right side (St5) versus the left side. One may speculate that a rightsided decrease in blood/O2 supply might occur in E and that this decrease may set a limit to the fuel/O2 consumption observed in the right side, thereby bringing E to a halt. Indeed, in ME (Figure 5), the base mitochondrial metabolism (St1) seems to be balanced between the 2 hemispheres, nevertheless, addition of ‘‘fuel’’ or ADP (St2-3) shows that the right side continues to possess the potentials for a more intense metabolism compared to the left. Also, the aforementioned potential left-sided inhibition (in E St2) does not seem to act in ME. The St4 results indicate that in ME, balanced base metabolism (St1) is also accompanied by balanced downregulatory processes (St4) in the 2 hemispheres. The St5 values suggest that blood/O2 supply in the hypothalamic sides has not changed compared to E. In DE (Figure 6), right-sided dominance was apparent (St1), which could not be further increased by the addition of P þ M and/or ADP (St2-3). We currently do not know the explanation of this phenomenon; however, increased downregulation (St4) in the right side was also observed that, with lower residual O2 left in the right side (St5), may play a role in equalizing the left–right balance by the onset of EP. Mitochondrial respiration measurements in hypothalamic sides of intact males showed nearly equal (no significant differences found) mrr values for both sides (data not shown). Thus, results indicate that hypothalamic asymmetry, at least regarding mitochondrial metabolism, exists only in females, but not in males.
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Figure 5. Left–right share in hypothalamic asymmetry in metestrus (ME). Base mitochondrial metabolism (St1) seems to be balanced between the 2 hemispheres, nevertheless, addition of ‘‘fuel’’ or adenosine diphosphate (ADP; St2-3) shows that the right side continues to possess the potentials for a more intense metabolism compared to the left. Also, the potential left-sided inhibition (in E St2) does not seem to act in ME. The St4 results indicate that in ME, balanced base metabolism (St1) is also accompanied by balanced downregulatory processes (St4) in the 2 hemispheres. The St5 values suggest that blood/O2 supply in the hypothalamic sides has not changed compared to E. St1 indicates state 1; St2, state 2; St3, state 3; St4; state 4.
Figure 6. Left–right share in hypothalamic asymmetry in diestrus (DE). Right-sided dominance was apparent (St1), which could not be further increased by the addition of P þ M and/or adenosine diphosphate (ADP; St2-3). We currently do not know the explanation of this phenomenon; however, increased downregulation (St4) in the right side was also observed that, with lower residual O2 left in the right side (St5), may play a role in equalizing the left–right balance by the onset of EP. St1 indicates state 1; St2, state 2; St3, state 3; St4; state 4.
Conclusion Histologically, the hypothalamus comprises like-named nuclei with symmetrical localization on the 2 sides of the third ventricle. Although early studies indicated the possibility of functional lateralization of the hypothalamus, those findings have been ignored by successive hypothalamic research that
Figure 7. Schematic of the possible structural basis of the hypothalamic metabolic lateralization. In female rats, an estrus phase-dependent and dynamically changing metabolic asymmetry was observed with either right- or left-sided dominance during the estrous cycle and in different experimental conditions. These dynamic changes suggest that certain mechanisms may exist that enable the dominant functions of one of the sides with the simultaneous inhibition of the contralateral side. Such a ‘‘give-or-take’’ mechanism assumes that there is some kind of interconnection between the hypothalamic hemispheres through which the stimulation of one side is functionally synchronized with the simultaneous inhibition of the contralateral side. The scheme summarizes the potential information channels between the 2 hypothalamic hemispheres through which the functional synchronization of the sided functions could be controlled. These include one or more of the following: (1) anatomical interconnection through the interthalamic adhesion and/or (2) the infundibular region; and (3) humoral interconnection through the lateral evaginations of the third ventricle; possibly an asymmetric sympathetic inhibition. We termed the hypothesized synchronizing mechanism ‘‘temporal, functional bilateral synchronization.’’
continued to consider the hypothalamus as an unpaired midline structure. Our present results, however, indicate that there is a sidedness in female, but not in male, hypothalamic functions, and because of its dependence on the estrous phase, it is most likely related to the regulation of GnRH secretion/release. These observations also raise the possibility that future consideration of hypothalamic functional laterality in hypothalamus research could lead to radically new observations regarding probably all known hypothalamic functions. A hypothesized structural basis of the mechanism that enables the stimulation of a hypothalamic side and the simultaneous contralateral inhibition is shown in Figure 7.
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Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: OTKA (Hungarian Scientific Research Fund) 81745 to Attila Zsarnovszky, OTKA 72186 to Tibor Bartha, OTKA 104982 to Tibor Bartha, and TAMOP (Social Renewal Operational Programme) 4.2.2. b-10/12010-0011 to David Sandor Kiss.
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