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Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αβγ) are linked to hibernating state in hamsters.

Alò R, Avolio E, Di Vito A, Carelli A, Facciolo RM, Canonaco M - BMC Neurosci (2010)

Bottom Line: At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe) and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%).We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster.The identification of a brain regional distribution pattern of distinct GABAAR subunit combinations may prove to be very useful for highlighting GABAergic mechanisms functioning at least during the different physiological states of hibernators and this may have interesting therapeutic bearings on neurological sleeping disorders.

View Article: PubMed Central - HTML - PubMed

Affiliation: Comparative Neuroanatomy Laboratory, Ecology Department, University of Calabria, Ponte Pietro Bucci, 87030 Arcavacata di Rende, Cosenza, Italy. canonaco@unical.it

ABSTRACT

Background: The structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR) is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR) tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB) and hibernating (HIB) hamsters. Attempts were made to identify the type of αβγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators.

Results: Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p < 0.01) prevalence of α1 ratio (over total α subunits considered in the present study) in the medial preoptic area (MPOA) and arcuate nucleus (Arc) of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe) and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%). Regarding the other two subunits (β and γ), elevated β3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for β3 and γ2 during hibernating conditions.

Conclusion: We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional distribution pattern of distinct GABAAR subunit combinations may prove to be very useful for highlighting GABAergic mechanisms functioning at least during the different physiological states of hibernators and this may have interesting therapeutic bearings on neurological sleeping disorders.

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Competition curves of [3H] Ro 15-1788 in the Sch of HIB and NHIB hamsters. Displacement curves of [3H] Ro 15-1788 (mean % of total binding ± s.e.m) showing the differing binding capacities in the suprachiasmatic nucleus (Sch) of a) HIB and b) NHIB hamsters. Competition study was carried out in the presence of different concentration (500 nM-1 nM) of α1 (zolpidem, white square) and α2 (flunitrazepam, black triangle) agonists plus α4 antagonist (Ro 15-4513, black square) and α5 inverse agonist, (Ry 080, white circle) as described in "Materials and Methods". Each point represents the mean of five separate tests.
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Figure 1: Competition curves of [3H] Ro 15-1788 in the Sch of HIB and NHIB hamsters. Displacement curves of [3H] Ro 15-1788 (mean % of total binding ± s.e.m) showing the differing binding capacities in the suprachiasmatic nucleus (Sch) of a) HIB and b) NHIB hamsters. Competition study was carried out in the presence of different concentration (500 nM-1 nM) of α1 (zolpidem, white square) and α2 (flunitrazepam, black triangle) agonists plus α4 antagonist (Ro 15-4513, black square) and α5 inverse agonist, (Ry 080, white circle) as described in "Materials and Methods". Each point represents the mean of five separate tests.

Mentions: In the present it was our intention to identify and establish the order of specific α-containing neuronal fields on the basis of their affinity levels characterizing some of the major hypothalamic areas during either HIB or NHIB states of our hamster model. Indeed, the labeling of the different hypothalamic sections with the radioligand [3H] Ro 15-1788 in the presence of distinct α subunit drugs and namely α1 (zolpidem) and α2 (flunitrazepam) agonists, plus α4 (Ro 15-4513) antagonist as well as a α5 (Ry 080) inverse agonist supplied a heterogeneous distribution pattern. In particular the results of the preliminary study, which confirmed previously published results [25], tended to point out that it was mainly α1 and α2 subunits of HIB (Figure 1a) and NHIB (Figure 1b), respectively, supplying greater high affinity type of characteristic as shown by their varying binding affinities and Bmax going from a high affinity range of 9.31 × 10-2 nM (327 fmol/mg protein) to 2.47 × 10-1 nM (404 fmol/mg protein) for these corresponding subunits. Such a relationship was also characterized by lower type of binding affinities of α4-containing sites and precisely 51.67 nM (Bmax = 215 fmol/mg protein) for NHIB hamsters while an affinity of 356.13 nM (Bmax = 393 fmol/mg protein) was obtained for hibernators (check their order in Figures 1a,b).


Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αβγ) are linked to hibernating state in hamsters.

Alò R, Avolio E, Di Vito A, Carelli A, Facciolo RM, Canonaco M - BMC Neurosci (2010)

Competition curves of [3H] Ro 15-1788 in the Sch of HIB and NHIB hamsters. Displacement curves of [3H] Ro 15-1788 (mean % of total binding ± s.e.m) showing the differing binding capacities in the suprachiasmatic nucleus (Sch) of a) HIB and b) NHIB hamsters. Competition study was carried out in the presence of different concentration (500 nM-1 nM) of α1 (zolpidem, white square) and α2 (flunitrazepam, black triangle) agonists plus α4 antagonist (Ro 15-4513, black square) and α5 inverse agonist, (Ry 080, white circle) as described in "Materials and Methods". Each point represents the mean of five separate tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2944354&req=5

Figure 1: Competition curves of [3H] Ro 15-1788 in the Sch of HIB and NHIB hamsters. Displacement curves of [3H] Ro 15-1788 (mean % of total binding ± s.e.m) showing the differing binding capacities in the suprachiasmatic nucleus (Sch) of a) HIB and b) NHIB hamsters. Competition study was carried out in the presence of different concentration (500 nM-1 nM) of α1 (zolpidem, white square) and α2 (flunitrazepam, black triangle) agonists plus α4 antagonist (Ro 15-4513, black square) and α5 inverse agonist, (Ry 080, white circle) as described in "Materials and Methods". Each point represents the mean of five separate tests.
Mentions: In the present it was our intention to identify and establish the order of specific α-containing neuronal fields on the basis of their affinity levels characterizing some of the major hypothalamic areas during either HIB or NHIB states of our hamster model. Indeed, the labeling of the different hypothalamic sections with the radioligand [3H] Ro 15-1788 in the presence of distinct α subunit drugs and namely α1 (zolpidem) and α2 (flunitrazepam) agonists, plus α4 (Ro 15-4513) antagonist as well as a α5 (Ry 080) inverse agonist supplied a heterogeneous distribution pattern. In particular the results of the preliminary study, which confirmed previously published results [25], tended to point out that it was mainly α1 and α2 subunits of HIB (Figure 1a) and NHIB (Figure 1b), respectively, supplying greater high affinity type of characteristic as shown by their varying binding affinities and Bmax going from a high affinity range of 9.31 × 10-2 nM (327 fmol/mg protein) to 2.47 × 10-1 nM (404 fmol/mg protein) for these corresponding subunits. Such a relationship was also characterized by lower type of binding affinities of α4-containing sites and precisely 51.67 nM (Bmax = 215 fmol/mg protein) for NHIB hamsters while an affinity of 356.13 nM (Bmax = 393 fmol/mg protein) was obtained for hibernators (check their order in Figures 1a,b).

Bottom Line: At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe) and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%).We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster.The identification of a brain regional distribution pattern of distinct GABAAR subunit combinations may prove to be very useful for highlighting GABAergic mechanisms functioning at least during the different physiological states of hibernators and this may have interesting therapeutic bearings on neurological sleeping disorders.

View Article: PubMed Central - HTML - PubMed

Affiliation: Comparative Neuroanatomy Laboratory, Ecology Department, University of Calabria, Ponte Pietro Bucci, 87030 Arcavacata di Rende, Cosenza, Italy. canonaco@unical.it

ABSTRACT

Background: The structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR) is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR) tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB) and hibernating (HIB) hamsters. Attempts were made to identify the type of αβγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators.

Results: Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p < 0.01) prevalence of α1 ratio (over total α subunits considered in the present study) in the medial preoptic area (MPOA) and arcuate nucleus (Arc) of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe) and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%). Regarding the other two subunits (β and γ), elevated β3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for β3 and γ2 during hibernating conditions.

Conclusion: We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional distribution pattern of distinct GABAAR subunit combinations may prove to be very useful for highlighting GABAergic mechanisms functioning at least during the different physiological states of hibernators and this may have interesting therapeutic bearings on neurological sleeping disorders.

Show MeSH