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GABAA receptors are located in cholinergic terminals in the nucleus pontis oralis of the rat: Implications for REM sleep control
brain research 1543 (2014) 58–64
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Research Report
GABAA receptors are located in cholinergic terminals in the nucleus pontis oralis of the rat: Implications for REM sleep control Chang-Lin Lianga,b, Gerald A. Marksa,b,n a
Department of Veterans Affairs, North Texas Health Care System, Dallas, TX 75216, USA Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
b
ar t ic l e in f o
abs tra ct
Article history:
The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as
Accepted 10 October 2013
a rapid eye movement (REM) sleep induction zone. Microinjection of GABAA receptor
Available online 17 October 2013
antagonists into PnO induces a long lasting increase in REM sleep, which is similar to that
Keywords: GABAA receptor subunit Immunohistochemistry Laser scanning confocal microscopy Pontine reticular formation REM sleep induction zone Vesicular transporter
produced by cholinergic agonists. We previously showed that this REM sleep-induction can be completely blocked by a muscarinic antagonist, indicating that the REM sleep-inducing effect of GABAA receptor antagonism is dependent upon the local cholinergic system. Consistent with these findings, it has been reported that GABAA receptor antagonists microdialyzed into PnO resulted in increased levels of acetylcholine. We hypothesize that GABAA receptors located on cholinergic boutons in the PnO are responsible for the REM sleep induction by GABAA receptor antagonists through blocking GABA inhibition of acetylcholine release. Cholinergic, varicose axon fibers were studied in the PnO by immunofluorescence and confocal, laser scanning microscopy. Immunoreactive cholinergic boutons were found to be colocalized with GABAA receptor subunit protein γ2. This finding implicates a specific subtype and location of GABAA receptors in PnO of rat in the control of REM sleep. Published by Elsevier B.V.
1.
Introduction
The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as a rapid eye movement (REM) sleep induction zone because of the increased REM sleep following the local intracerebral injection of a variety of
different neurotransmitter-receptor ligands (Ahnaou et al., 1999, 2000; Bourgin et al., 1999, 1995, 1997; Marks and Birabil, 1998; Marks et al., 2003). One factor controlling REM sleep in PnO is the number of functional GABA-type A receptors (GABAAR). In rat, small volume injections of GABAAR antagonists directly into PnO produce a long-lasting increase in REM
Abbreviations: ACh, acetylcholine; BZ, benzodiazepine; GABAAR, type-A GABA receptor; GAD67, glutamic acid decarboxylase-67; KCC2, potassium-chloride cotransporter-2; MAP2, microtubule-associated protein 2; PB, phosphate buffer; PnO, oral pontine reticular formation; REM, rapid eye movement; VAChT, vesicular acetylcholine transporter; VGAT, vesicular GABA transporter n Corresponding author at: Department of Veterans Affairs, North Texas Health Care System Research, VA Medical Center, MC# 151, Dallas 4500 South Lancaster Road, Dallas, TX 75216, USA. Fax: þ1 214 462 4966. E-mail address: [email protected] (G.A. Marks). 0006-8993/$ - see front matter Published by Elsevier B.V. http://dx.doi.org/10.1016/j.brainres.2013.10.019
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sleep (Marks et al., 2008; Sanford et al., 2003). GABAARs are abundant in the reticular formation (Pirker et al., 2000) and probably serve a number of roles in different functions ascribed to the PnO. The subset of GABAARs specifically involved in REM sleep control is unknown. The nature of the REM sleep induction by GABAAR antagonists in rat is very similar to that produced by cholinergic agonists injected into the PnO (Bourgin et al., 1995; Marks and Birabil, 1998). This led us to demonstrate that the muscarinic antagonist, atropine, at doses not affecting REM sleep, completely blocked the GABAAR antagonist induction (Marks et al., 2008). This indicates that GABA's action is dependent upon the local cholinergic system. In addition, GABAAR antagonists microdialyzed into PnO of cat, or mouse, resulted in increased levels of ACh (Vazquez and Baghdoyan, 2004; Flint et al., 2010). Taken together, these findings are consistent with reduction in GABA's action in PnO to increase REM sleep by promoting acetylcholine release from local cholinergic terminals. This then results in an atropine-sensitive, cholinergic induction of REM sleep. Several, mechanisms could underlie the actions of GABAAR antagonists in PnO on local cholinergic transmission. These include disinhibition of neurons in PnO with excitatory projections to the cholinergic neurons innervating PnO or disinhibition of PnO neurons with local action on cholinergic terminals promoting acetylcholine release. Yet another alternative is the direct action of injected GABAAR antagonists on cholinergic terminals in PnO promoting acetylcholine release. Presynaptic GABAARs in multiple brain loci have been shown to have a role in control of release of a variety of neurotransmitters, including acetylcholine (ACh; reviewed in MacDermott et al., 1999). To the extent this latter mechanism is operating in PnO, the GABAARs mediating REM sleep control should be located in cholinergic axon fibers and varicosities in PnO. Here we test the hypothesis that GABAARs are present in cholinergic, presynaptic terminals in the rat PnO. We found that many varicosities labeled with the cholinergic marker, vesicular acetylcholine transporter protein (VAChT;Gilmor et al.,1996), also colocated immunoreactivity for the γ2 subunit of the GABAAR. This supports the concept of direct GABAergic modulation of ACh release in the PnO as a mechanism of REM sleep control.
2.
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distribution of the γ2-puncta lacked an aggregation to somata plasma membranes, consistent with the description of γ2 subunit expressed in processes in PnO rather than somata (Pirker et al., 2000). Utilizing an antibody against microtubuleassociated protein 2 (MAP2) to label dendrites (De Camilli et al., 1984), γ2-puncta was found apposed to dendritic membranes, but no γ2 label was observed in the dendrites (Fig. 1). This indicates GABAARs containing γ2 subunits may have a distribution, in PnO, restricted to axon fibers. In support of our hypothesis, we found numerous VAChTlabeled varicosities localizing the γ2 subunit. Double-labeled varicosities had no obvious relationship to size of the varicosity. A total of 867 VAChT-labeled varicosities were counted in PnO of four rats. Of these, 70 were identified as co-labeled for the γ2 subunit. This yielded a mean7SEM of 8.270.71% doublelabeled cholinergic varicosities (Table 1). GABAAR γ2 subunit, punctate labeling identical to that found in varicosities, also was observed in the absence of VAChT labeling. A total of 207 were counted in the fields sampled. With 70 found co-labeled with VAChT, 34.2%74.16% of total γ2-labeled puncta in PnO were located in cholinergic varicosities (Table 1). Including a third label for the processes of GABAergic neurons revealed many instances in which double-labeled, VAChT/γ2 varicosities were found apposed, with no space between the elements, to a varicose axon fiber labeled for glutamic acid decarboxylase-67 (GAD67) or vesicular GABA transporter (VGAT; Fig. 2A–F). This is highly suggestive of synaptic contact. In as much as VGAT is a presynaptic marker and cholinergic boutons express GABAARs, it is consistent with cholinergic boutons being postsynaptic to GABAergic axons. Fig. 3A–D shows a clear example of a single, co-labeled, VAChT/γ2 varicosity apposed by two varicosities labeled for VGAT.
3.
Discussion
GABAARs are ligand-gated Cl ion channels structurally assembled from five individual protein subunits, which include combinations of α1–6, β1–3, γ1–3, δ, ε, π and θ (Pirker et al., 2000). GABAARs consist of a large number of receptor-subtypes based
Results
VAChT labeled varicosities appeared with moderately high density in PnO. When axon fibers were visible, most coursed within the plane of section where they branched frequently and expressed a high number of varicosities. Most of these varicosities resembled beads on a string, which we interpret as boutons en passant. To a lesser extent, boutons terminaux also were observed based on their terminal position along an axon fiber. Some varicosities appeared to be apposed to somata, but the majority did not. Cholinergic varicosities were found in a wide range of sizes from approximately 1 to 5 μm in diameter. Immuno-label for the γ2 subunit had a punctate appearance consistent with being associated with clustered GABAARs, as has been described for the γ2 subunit (Essrich, et al., 1998). The
Fig. 1 – Fluorescence confocal image of a representative field in PnO showing MAP2-labeled dendrites (red) and punctate label for the γ2 GABAAR-subunit (blue). The γ2-label is not found inside dendrites (arrows). The MAP2/γ2 label indicated by arrow head is not colocalized in the z-dimension.
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Table 1 – Sampled counts of cholinergic varicsities and γ2 GABAAR-subunit label. Subj.
Total
Total
Total double
% Double
% Double
Rat ♯1 Rat ♯2 Rat ♯3 Rat ♯4 Mean SEM
VAChTþ 263 201 249 154 216.75 721.45
γ 49 46 56 56 51.75 72.19
VAChTþ/γ 16 20 22 12 17.5 71.92
VAChT 6.08 9.95 8.84 7.79 8.17 70.71
γ2 32.65 43.48 39.29 21.43 34.21 74.16
Counts are the totals of fields in PnO on two sections from each rat. % Double VACHT, percentage of total VAChT labeled varicosities colabled for the γ2 subunit. % Double γ2, percentage of total punctate γ2 label found in VAChT labeled varicosities. SEM, standard error of the mean.
Fig. 2 – Fluorescence confocal image of representative double-labeled varicosities in PnO co-expressing VAChT (blue) and the γ2 GABAAR-subunit (red) apposed by varicosities labeled for VGAT (green). (A)–(F) a single field in PnO showing individual, (A)–(C) and combinations of color channels, (D)–(F). (D) VAChT/γ2; and (E), VAChT/VGAT; (F) all three channels merged. Scale bar in F, 5 μm.
on their subunit composition and this gives rise to a multiplicity of functional and pharmacological properties (reviewed in Fritschy and Brünig, 2003 and Olsen and Sieghart, 2009). Individual subunits have been visualized utilizing immunohistochemistry (Fritschy et al., 1992) and most of the subunits tested were observed to be expressed in PnO (Pirker et al., 2000). The presence of a subunit is putative evidence for the expression of functional GABAAR-subtypes containing that subunit. Colocalization of VAChT labeled varicosities and the γ2 GABAAR-subunit in the PnO is consistent with direct presynaptic control over ACh release by GABA. γ2 subunit immunoreactivity has been reported in processes in PnO with moderate density (Pirker et al., 2000). This mechanism is supported further by local blockade of GABAARs in PnO of cat and mouse resulting in increased levels of ACh (Flint et al., 2010; Vazquez and Baghdoyan, 2004), suggesting that control by GABA is inhibitory to ACh-release in PnO. Cholinergic agonists and GABAAR antagonists injected into the PnO of
rat induced increase in REM sleep with very similar characteristics and both drug-inductions were blocked by the muscarinic receptor antagonist atropine (Marks et al., 2008). Thus, the mechanism of GABAAR antagonism is dependent upon the function of local cholinergic receptors. The presence of GABAARs on cholinergic boutons in PnO favors the direct action of GABAAR blockade on these presynaptic terminals to increase ACh release and result in a cholinergic REM sleep induction mediated through muscarinic receptors in PnO. Additional mechanisms of GABA's action on REM sleep cannot be excluded. GABAARs are ubiquitously expressed in the PnO (Pirker et al., 2000). Data presented here support the conclusion that specific GABAARs mediating REM sleep-induction by GABAAR antagonists are located on boutons of cholinergic axon fibers and, at least in part, include a subtype containing the γ2 subunit. The presynaptic mechanism of GABAAR inhibition of AChrelease in PnO is not known. Reported paucity of expression of
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Fig. 3 – Example of a single, double-labeled, cholinergic varicosity in PnO (VAChT) colabeled for the γ2 subunit and clearly apposed by two VGAT labeled varicosities (arrows). Color assignments same as in Fig. 1. (A)–(C), individual color channels and (D) all three channels merged. Scale bar in (D), 5 μm.
the main Cl extrusion system, potassium-chloride cotransporter-2 (KCC2), in neuronal axons favors a depolarizing action at these sites (reviewed in Blaesse et al., 2009). We have observed a lack of KCC2-immunoreactivity in VAChT-labeled varicose fibers in the PnO (Liang and Marks, 2011). GABAAR-mediated depolarization could result in inhibiting ACh-release in PnO through a variety of mechanisms, including inactivation of Naþ channels, Ca2þ channels, and shunting of current (reviewed in Kullmann et al., 2005). These actions would result in reduced exocytotic release of ACh, possibly by blockade of action potential propagation. The lack of expression of γ2-puncta in membranes of somata and dendrites is consistent with γ2 subunit in PnO selectively expressed in axon fibers. This indicates that the major role of GABAARs containing γ2 subunits in PnO is to function in the presynaptic modulation of the release of neurotransmitters. Greater than one-third of the γ2-counts were found in cholinergic boutons consistent with ACh being one neurotransmitter system being modulated by GABA. Which other neurotransmitter systems in PnO express γ2 in their axons and what functions they may have remains to be explored. VAChT-labeled varicosities in PnO, however, were found to colocalize γ2 subunit labeling with only about an 8% incidence. This could occur if γ2 subunit-containing receptor expression were dynamic and drop below the level of detection.
Additionally, multiple GABAAR-subtypes may be distributed among the cholinergic varicosities and not all subtypes contain the γ2 subunit. Alternatively, GABAARs may not be present at every cholinergic bouton and GABA inhibition of ACh release in the PnO may not be acting at each individual presynaptic site of ACh release. One possibility is that cholinergic boutons in PnO containing γ2 subunits are functionally distinct, preferentially providing ACh access to postsynaptic muscarinic receptors specifically involved in REM sleep control. This may include a specific source of cholinergic innervation of PnO or subpopulations from specific sources, for example, the minor proportion of putative mesopontine cholinergic neurons that may provide input to PnO whose activity is selective to REM sleep (Datta and Siwek, 2002; Kayama et al., 1992). Another possibility relates to the nature of the extensive varicose axonal arbors of cholinergic neurons in PnO. These axons could be subjected to GABAmediated depolarization at selective sites along the axon and, through a shunting of current, result in blockade of action potential propagation to distal branches. By this mechanism, an entire region, such as PnO, could effectively be deprived of cholinergic innervation that requires impulse-dependent release. This latter mechanism is consistent with the state-related activity of the majority of mesopontine cholinergic neurons
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discharging at their highest rates in wake and REM sleep (Datta and Siwek, 2002; Kayama et al., 1992). Based on injections of retrograde tracers, the majority of these cholinergic neurons projecting to the pontine reticular formation also have rostral projections including the thalamus (Semba et al., 1990). ACh levels measured by microdialysis in the thalamus are high during both REM sleep and wake compared to NREM sleep (Williams et al., 1994), yet, ACh levels measured in cat pontine reticular formation are the highest in REM sleep alone compared to wake and NREM sleep (Kodama et al., 1990). GABA could act locally through a mechanism blocking impulse-dependent release of ACh in the PnO selectively during wakefulness. Natural reduction of this GABA inhibition during REM sleep, or local pharmacological antagonism of GABAARs, would restore a relationship between activity of cholinergic neurons and ACh release. We suggest it is the increase in ACh release during wakefulness produced by GABAAR antagonists in PnO that results in induction of REM sleep. We identified γ2 as a subunit of at least one GABAAR subtype localized to cholinergic boutons in the PnO. Other subunits constituting these receptors are yet unknown. Nonetheless, just knowledge of γ2 as a subunit permits certain inferences. γ2 containing subtypes are associated with receptors possessing benzodiazepine (BZ) binding sites (Kucken et al., 2000). Additional support for the role of BZ binding sites is our recent finding of induction of REM sleep with the local application of a BZ site inverse agonist in PnO that is also blocked by atropine (Nguyen et al., 2013). Conversely, agonists at the BZ site potentiate the action of GABA and commonly result in reduced REM sleep when prescribed as hypnotics (Lancel, 1999). This is consistent with action of these drugs at receptors in PnO in which antagonizing GABA function increases REM sleep and potentiating function reduces REM sleep (Flint et al., 2010; Sanford, et al., 2003; Vanini and Baghdoyan, 2013). The γ2 subunit is thought to be involved in the mechanism of targeting some receptor subtypes to synaptic locations (Essrich et al., 1998). We have observed double-labeled VAChT/γ2 varicosities in close apposition to varicosities labeled with VGAT (Figs. 2 and 3) and GAD (not shown). This observation is consistent with the GABAARs on cholinergic boutons in rat PnO being postsynaptic to a specific GABAergic innervation. Identifying the source, or sources, of this GABAergic innervation presynaptic to cholinergic terminals in PnO should reveal a critical, up-stream mechanism of REM sleep control. GABAergic neurotransmission in the PnO is likely to constitute a natural mechanism of REM sleep control. GABA
levels, studied with in vivo microdialysis in the pontine reticular formation of the cat, were found to undergo significant alterations with spontaneous changes in the states of arousal (Vanini et al., 2011). Levels of GABA were significantly lower during natural REM sleep compared to both NREM sleep and wakefulness. This is the reciprocal of the relationship found for ACh, which is significantly higher during REM sleep compared to both NREM sleep and wakefulness (Kodama et al., 1990). This reciprocal relationship is consistent with GABAergic inhibition of ACh release in PnO and the central role both neurotransmitters play in control of REM sleep.
4.
Conclusion
GABAARs are expressed in cholinergic varicose axon fibers within the pontine reticular formation's REM sleep induction zone supporting the direct action of GABA on cholinergic boutons. Synaptic release of GABA during wakefulness would inhibit ACh release while reduced GABA-action would result in increased ACh release promoting REM sleep.
5.
Experimental procedure
All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the local Institutional Animal Care and Use Committee at the Dallas VA. All efforts were made to minimize the number of animals used and their suffering.
5.1.
Tissue collection
Four adult male, Long-Evans Hooded rats (Harlan, Indianapolis, IN, USA) weighing between 180 and 250 g were housed under standard conditions on a 12/12 h light/dark schedule. At between 3 and 5 h after lights-on, rats were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and then perfused through the ascending aorta with 30 ml heparinized saline and 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 30 min. The brains were removed, blocked in the coronal plane, post-fixed in the same fixative for 1 h and cryoprotected by immersion in 20% sucrose in PB. Serial, coronal sections, 20 μm-thick were cut on a freezing microtome (SM2000R, Leica, Nussloch, Germany) through the pons, and collected in 0.1 M PB, with 0.005% sodium azide. The region of interest was the caudal aspect of the PnO, based on the high degree of effectiveness of
Table 2 – Primary antibodies used in the study. Antigen
Species raised in
Source and code
Dilution
Specificity reference
VAChT
Goat
Chemicon, AB1578 Temecula, CA, USA
1:2000
γ2
Rabbit
PhosphoSolutions 832-GG2C Auroray, CO, USA
1:500
VGAT GAD67
Mouse Mouse
Synaptic Systems 131 011 Göttingen, Germanyf Chemicon/Millipore MAB5406 Temecula, CA, USA
1:1000 1:1000
MAP2
Chicken
Novus, NB300–213 Littleton, CO, USA
1:10,000
Arvidsson et al. (1997) Henny and Jones (2006) Sperk et al. (1997) Pirker et al. (2000) Takamori et al. (2000) Verea et al. (2005) Watanabe et al. (2005) Li et al. (2010)
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GABAAR antagonists to induce REM sleep at this site (Marks et al., 2008). This was defined as the area of the pontine reticular formation included in the 0.5 mm caudal to the decussation of the superior cerebellar peduncle, approximately 8.3–8.8 mm posterior to the bregma suture (Paxinos and Watson, 1997).
5.2. Immunohistochemistry, microscopy and image acquisition Free-floating sections were primarily blocked with 3% normal donkey serum and 0.3% Triton X-100 in PBS for 60 min and then incubated in a mixture of primary antibodies for VAChT and γ2 or MAP2 and γ2 (double staining), or VAChT, γ2 and, GABAergic neuronal markers, vesicular GABA transporter (VGAT) or glutamic acid decarboxylase-67 (GAD67;triple staining) for 18–20 h at 4 1C (see Table 2). After washing with PBS, sections were incubated with fluorescent-tagged (Cy2, Cy3 and Cy5) secondary antibodies for 3 h (Jackson Immuno Research Labs, West Grove, PA, USA). After 30 min wash with PBS, sections were mounted and cover-slipped with mounting medium containing glycerin and phenylenediamine. Negative controls for antibody labeling indicated a lack of specific staining when primary or secondary antibodies were omitted. Confocal microscopy was performed to examine the relationship between VAChT and γ2 positive varicosities as well as the relationships among VAChT, γ2 and, VGAT or GAD67 positive varicosities in PnO. The relationship of γ2 to dendrites was assessed using MAP2 labeling (De Camilli et al., 1984). Simultaneous epifluorescence (Cy2 and Cy3, or Cy2, Cy3 and Cy5) images were obtained using a laser-scanning confocal microscope (LSM510-META, Zeiss, Göttingen, Germany) with a 100 objective (oil; N.A., 1.4; pin hole, 1 Airy Unit all channels). Excitation was provided by three lasers with excitation wave lengths of 488, 543, and 633 nm. Laser-scanning sectioning in the Z plane was performed using multitrack scanning. Two coronal sections, separated by 250 μm, were randomly chosen from each of the four rats. Starting 2 μm below the surface of each section, a z-stack of at least 6 optical slices was obtained in 0.33 μm steps. While viewing fluorescence emission solely from the VAChT-label, an xy-field was chosen around the center of the PnO to insure a sufficient number of cholinergic varicosities for counting. Colocalization was assessed by viewing all immuno-labels in the field on adjacent Z sections. No significant differences in counts were found between the pairs of sections and they were combined for total VAChT, γ2 and their colocalization. Additional sections were chosen to determine the relationship of γ2 with MAP2 labeling. Confocal images were imported into Photoshop software (CS4, Adobe, San Jose, CA, USA) for composition of merged images and global adjustments of brightness and contrast. Some confocal images were imported into Volocity software (v. 5.2, Improvision, Waltham, MA, USA) for deconvolution and 3-D reconstruction.
Acknowledgments This work was supported by Department of Veterans Affairs Merit Review. We thank Ginger (Jinghua) Zhou for her expert assistance in the histological preparation of brain tissue.
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r e f e r e nc e s
Ahnaou, A., Basille, M., Gonzalez, B., Vaudry, H., Hamon, M., Adrien, J., Bourgin, P., 1999. Long-term enhancement of REM sleep by the pituitary adenylyl cyclase-activating polypeptide (PACAP) in the pontine reticular formation of the rat. Eur. J. Neurosci. 11, 4051–4058. Ahnaou, A., Laporte, A.M., Ballet, S., Escourrou, P., Hamon, M., Adrien, J., Bourgin, P., 2000. Muscarinic and PACAP receptor interactions at pontine level in the rat: significance for REM sleep regulation. Eur. J. Neurosci. 12, 4496–4504. Arvidsson, U., Riedl, M., Elda, R., Meister, B., 1997. Vesicular acetylcholine transporter (VAChT) protein: a novel and unique marker for cholinergic neurons in the central and peripheral nervous system. J. Comp. Neurol. 378, 454–467. Blaesse, P., Airaksinen, M.S., Rivera, C., Kaila, K., 2009. Cationchloride cotransporters and neuronal function. Neuron 61, 820–838. Bourgin, P., Ahnaou, A., Laporte, A.M., Hamon, M., Adrien, J., 1999. Rapid eye movement sleep induction by vasoactive intestinal peptide infused into the oral pontine tegmentum of the rat may involve muscarinic receptors. Neuroscience 89, 291–302. Bourgin, P., Escourrou, P., Gaultier, C., Adrien, J., 1995. Induction of rapid eye movement sleep by carbachol infusion into the pontine reticular formation in the rat. NeuroReport 6, 532–536. Bourgin, P., Lebrand, C., Escourrou, P., Gaultier, C., Franc, B., Hamon, M., Adrien, J., 1997. Vasoactive intestinal polypeptide microinjections into the oral pontine tegmentum enhance rapid eye movement sleep in the rat. Neuroscience 77, 351–360. Datta, S., Siwek, D.F., 2002. Single cell activity patterns of pedunculopontine tegmentum neurons across the sleep–wake cycle in the freely moving rats. J. Neurosci. Res. 70, 611–621. De Camilli, P., Miller, P.E., Navone, F., Theurkauf, W.E., Vallee, R.B., 1984. Distribution of microtubule-associated protein 2 in the nervous system of the rat studied by immunofluorescence. Neuroscience 11, 819–846. Essrich, C., Lorez, M., Benson, J.A., Fritschy, J.-M., Lüscher, B., 1998. Postsynaptic clustering of GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. Nat. Neurosci. 1, 563–571. Flint, R.R., Chang, T., Lydic, R., Baghdoyan, H.A., 2010. GABAA receptors in the pontine reticular formation of C57BL6J mouse modulate neurochemical, electrographic, and behavioral phenotypes of wakefulness. J. Neurosci. 30, 12301–12309. Fritschy, J.-M., Benke, D., Mertens, S., Oertel, W.H., Bachi, T., Möhler, H., 1992. Five subtypes of type A γ-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies. Proc. Natl. Acad. Sci. USA 89, 6726–6730. Fritschy, J.-M., Brünig, I., 2003. Formation and plasticity of GABAergic synapses: physiological mechanisms and pathophysiological implications. Pharmacol. Ther. 98, 299–323. Gilmor, M.L., Nash, N.R., Roghani, A., Edwards, R.H., Yi, H., Hersch, S.M., Levey, A.I., 1996. Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles. J. Neurosci. 16, 2179–2190. Henny, P., Jones, B.E., 2006. Vesicular glutamate (VgluT), GABA (VGAT), and acetylcholine (VAChT) transporters in basal forebrain axon terminals innervating the lateral hypothalamus. J. Comp. Neurol. 496, 53–467. Kayama, Y., Ohta, M., Jodo, E., 1992. Firing of ‘possibly’ cholinergic neurons in the rat laterodorsal tegmental nucleus during sleep and wakefulness. Brain Res. 569, 210–220.
64
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Kodama, T., Takahashi, Y., Honda, Y., 1990. Enhancement of acetylcholine release during paradoxical sleep in the dorsal tegmental field of the cat brain stem. Neurosci. Lett 114, 277–282. Kucken, A.M., Wagner, D.A., Ward, P.R., Teissére, J.A., Boileau, A.J., Czajkowski, C., 2000. Identification of benzodiazepine binding site residues in the γ2 subunit of the γ-aminobutyric acidA receptor. Mol. Pharmacol. 57, 932–939. Kullmann, D.M., Ruiz, A., Rusakov, D.M., Scott, R., Semyanov, A., Walker, M.C., 2005. Presynaptic, extrasynaptic and axonal GABAA receptors in the CNS: where and why?. Prog. Biophys. Mol. Biol. 87, 33–46. Lancel, M., 1999. Role of GABAA receptors in the regulation of sleep: initial sleep responses to peripherally administered modulators and agonists. Sleep 22, 33–42. Li, Y., Serwanski, D.R., Miralles, C.P., Fiondella, C.G., LoTurco, J.J., Rubio, M.E., De Blas, A.L., 2010. Synaptic and non-synaptic localization of protocadherin-γC5 in the rat brain. J. Comp. Neurol. 518, 3439–3463. Liang C.-L., Marks, G.A., 2011. On expression of certain proteins in mesopontine cholinergic neurons at their soma and terminals in the pontine reticular formation. Program ♯505.12, Neuroscience Meeting Planner, Society for Neuroscience, Washington, DC, Online. MacDermott, A.B., Role, L.W., Siegelbaum, S.A., 1999. Presynaptic ionotropic receptors and the control of transmitter release. Annu. Rev. Neurosci. 22, 443–485. Marks, G.A., Birabil, C.G., 1998. Enhancement of rapid eye movement sleep in the rat by cholinergic and adenosinergic agonists infused into the pontine reticular formation. Neuroscience 86, 29–37. Marks, G.A., Sachs, O.W., Birabil, C.G., 2008. Blockade of GABA, type A, receptors in the rat pontine reticular formation induces rapid eye movement sleep that is dependent upon the cholinergic system. Neuroscience 156, 1–10. Marks, G.A., Shaffery, J.P., Speciale, S.G., Birabil, C.G., 2003. Enhancement of rapid eye movement sleep in the rat by actions at A1 and A2a adenosine receptor subtypes with a differential sensitivity to atropine. Neuroscience 116, 913–920. Nguyen, T.Q., Liang, C.-L., Marks, G.A., 2013. GABAA receptors implicated in REM sleep control express a benzodiazepine binding site. Brain Res. 1527, 131–140. Olsen, R.W., Sieghart, W., 2009. GABA A receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology 60, 243–260.
Paxinos, G., Watson, C., 1997. The Rat Brain in Stereotaxic Coordinates: Compact, 3rd Ed. Academic Press, San Diego (CD-ROM). Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W., Serk, G., 2000. GABAA receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101, 815–850. Sanford, L.D., Tang, X., Xiao, J., Ross, R.J., Morrison, A.R., 2003. GABAergic regulation of REM sleep in reticularis pontis oralis and caudalis in rats. J. Neurophysiol. 90, 938–945. Semba, K., Reiner, P.B., Fibiger, H.C., 1990. Single cholinergic mesopontine tegmental neurons project to both the pontine reticular formation and the thalamus in the rat. Neuroscience 38, 643–654. Sperk, G., Schwarzer, C., Tsunashima, K., Fuchs, K., Sieghart, W., 1997. GABAA receptor subunits in the rat hippocampus I: immunocytochemical distribution of 13 subunits. Neuroscience 80, 987–1000. Takamori, S., Riedel, D., Jahn, R., 2000. Immunoisolation of GABAspecific synaptic vesicles defines a functionally distinct subset of synaptic vesicles. J. Neurosci. 20, 4904–4911. Vanini, G., Wathen, B.L., Lydic, R., Baghdoyan, H.A., 2011. Endogenous GABA levels in the pontine reticular formation are greater during wakefulness than during rapid eye movement sleep. J. Neurosci. 31, 2649–2656. Vanini, G., Baghdoyan, H.A., 2013. Extrasynaptic GABAA receptors in rat pontine reticular formation increase wakefulness. Sleep 36, 337–343. Vazquez, J., Baghdoyan, H.A., 2004. GABAA receptors inhibit acetylcholine release in cat pontine reticular formation: implications for REM sleep regulation. J. Neurophysiol. 92, 2198–2206. Verea, E., Nácher, J., Blasco-Ibáñez, J.M., Gómez-Climen, M.A., Castillo-Gómez, E., Crespo, C., Martinez-Guijarro, F.J., 2005. PSA-NCAM expression in the rat medial prefrontal cortex. Neuroscience 136, 435–443. Watanabe, K., Kamiya, D., Nishiyama, A., Katayama, T., Nozaki, S., Kawasaki, H., Watanabe, Y., Mizuseki, K., Sasai, Y., 2005. Directed differentiation of telencephalic precursors from embryonic stem cells. Nature Neurosci. 8, 288–296. Williams, J.A., Comisarow, J., Day, J., Fibiger, H.C., Reiner, P.B., 1994. State-dependent release of acetylcholine in rat thalamus measured by in vivo microdialysis. J. Neurosci. 14, 5236–5242.
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Research Report
GABAA receptors are located in cholinergic terminals in the nucleus pontis oralis of the rat: Implications for REM sleep control Chang-Lin Lianga,b, Gerald A. Marksa,b,n a
Department of Veterans Affairs, North Texas Health Care System, Dallas, TX 75216, USA Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
b
ar t ic l e in f o
abs tra ct
Article history:
The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as
Accepted 10 October 2013
a rapid eye movement (REM) sleep induction zone. Microinjection of GABAA receptor
Available online 17 October 2013
antagonists into PnO induces a long lasting increase in REM sleep, which is similar to that
Keywords: GABAA receptor subunit Immunohistochemistry Laser scanning confocal microscopy Pontine reticular formation REM sleep induction zone Vesicular transporter
produced by cholinergic agonists. We previously showed that this REM sleep-induction can be completely blocked by a muscarinic antagonist, indicating that the REM sleep-inducing effect of GABAA receptor antagonism is dependent upon the local cholinergic system. Consistent with these findings, it has been reported that GABAA receptor antagonists microdialyzed into PnO resulted in increased levels of acetylcholine. We hypothesize that GABAA receptors located on cholinergic boutons in the PnO are responsible for the REM sleep induction by GABAA receptor antagonists through blocking GABA inhibition of acetylcholine release. Cholinergic, varicose axon fibers were studied in the PnO by immunofluorescence and confocal, laser scanning microscopy. Immunoreactive cholinergic boutons were found to be colocalized with GABAA receptor subunit protein γ2. This finding implicates a specific subtype and location of GABAA receptors in PnO of rat in the control of REM sleep. Published by Elsevier B.V.
1.
Introduction
The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as a rapid eye movement (REM) sleep induction zone because of the increased REM sleep following the local intracerebral injection of a variety of
different neurotransmitter-receptor ligands (Ahnaou et al., 1999, 2000; Bourgin et al., 1999, 1995, 1997; Marks and Birabil, 1998; Marks et al., 2003). One factor controlling REM sleep in PnO is the number of functional GABA-type A receptors (GABAAR). In rat, small volume injections of GABAAR antagonists directly into PnO produce a long-lasting increase in REM
Abbreviations: ACh, acetylcholine; BZ, benzodiazepine; GABAAR, type-A GABA receptor; GAD67, glutamic acid decarboxylase-67; KCC2, potassium-chloride cotransporter-2; MAP2, microtubule-associated protein 2; PB, phosphate buffer; PnO, oral pontine reticular formation; REM, rapid eye movement; VAChT, vesicular acetylcholine transporter; VGAT, vesicular GABA transporter n Corresponding author at: Department of Veterans Affairs, North Texas Health Care System Research, VA Medical Center, MC# 151, Dallas 4500 South Lancaster Road, Dallas, TX 75216, USA. Fax: þ1 214 462 4966. E-mail address: [email protected] (G.A. Marks). 0006-8993/$ - see front matter Published by Elsevier B.V. http://dx.doi.org/10.1016/j.brainres.2013.10.019
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sleep (Marks et al., 2008; Sanford et al., 2003). GABAARs are abundant in the reticular formation (Pirker et al., 2000) and probably serve a number of roles in different functions ascribed to the PnO. The subset of GABAARs specifically involved in REM sleep control is unknown. The nature of the REM sleep induction by GABAAR antagonists in rat is very similar to that produced by cholinergic agonists injected into the PnO (Bourgin et al., 1995; Marks and Birabil, 1998). This led us to demonstrate that the muscarinic antagonist, atropine, at doses not affecting REM sleep, completely blocked the GABAAR antagonist induction (Marks et al., 2008). This indicates that GABA's action is dependent upon the local cholinergic system. In addition, GABAAR antagonists microdialyzed into PnO of cat, or mouse, resulted in increased levels of ACh (Vazquez and Baghdoyan, 2004; Flint et al., 2010). Taken together, these findings are consistent with reduction in GABA's action in PnO to increase REM sleep by promoting acetylcholine release from local cholinergic terminals. This then results in an atropine-sensitive, cholinergic induction of REM sleep. Several, mechanisms could underlie the actions of GABAAR antagonists in PnO on local cholinergic transmission. These include disinhibition of neurons in PnO with excitatory projections to the cholinergic neurons innervating PnO or disinhibition of PnO neurons with local action on cholinergic terminals promoting acetylcholine release. Yet another alternative is the direct action of injected GABAAR antagonists on cholinergic terminals in PnO promoting acetylcholine release. Presynaptic GABAARs in multiple brain loci have been shown to have a role in control of release of a variety of neurotransmitters, including acetylcholine (ACh; reviewed in MacDermott et al., 1999). To the extent this latter mechanism is operating in PnO, the GABAARs mediating REM sleep control should be located in cholinergic axon fibers and varicosities in PnO. Here we test the hypothesis that GABAARs are present in cholinergic, presynaptic terminals in the rat PnO. We found that many varicosities labeled with the cholinergic marker, vesicular acetylcholine transporter protein (VAChT;Gilmor et al.,1996), also colocated immunoreactivity for the γ2 subunit of the GABAAR. This supports the concept of direct GABAergic modulation of ACh release in the PnO as a mechanism of REM sleep control.
2.
59
distribution of the γ2-puncta lacked an aggregation to somata plasma membranes, consistent with the description of γ2 subunit expressed in processes in PnO rather than somata (Pirker et al., 2000). Utilizing an antibody against microtubuleassociated protein 2 (MAP2) to label dendrites (De Camilli et al., 1984), γ2-puncta was found apposed to dendritic membranes, but no γ2 label was observed in the dendrites (Fig. 1). This indicates GABAARs containing γ2 subunits may have a distribution, in PnO, restricted to axon fibers. In support of our hypothesis, we found numerous VAChTlabeled varicosities localizing the γ2 subunit. Double-labeled varicosities had no obvious relationship to size of the varicosity. A total of 867 VAChT-labeled varicosities were counted in PnO of four rats. Of these, 70 were identified as co-labeled for the γ2 subunit. This yielded a mean7SEM of 8.270.71% doublelabeled cholinergic varicosities (Table 1). GABAAR γ2 subunit, punctate labeling identical to that found in varicosities, also was observed in the absence of VAChT labeling. A total of 207 were counted in the fields sampled. With 70 found co-labeled with VAChT, 34.2%74.16% of total γ2-labeled puncta in PnO were located in cholinergic varicosities (Table 1). Including a third label for the processes of GABAergic neurons revealed many instances in which double-labeled, VAChT/γ2 varicosities were found apposed, with no space between the elements, to a varicose axon fiber labeled for glutamic acid decarboxylase-67 (GAD67) or vesicular GABA transporter (VGAT; Fig. 2A–F). This is highly suggestive of synaptic contact. In as much as VGAT is a presynaptic marker and cholinergic boutons express GABAARs, it is consistent with cholinergic boutons being postsynaptic to GABAergic axons. Fig. 3A–D shows a clear example of a single, co-labeled, VAChT/γ2 varicosity apposed by two varicosities labeled for VGAT.
3.
Discussion
GABAARs are ligand-gated Cl ion channels structurally assembled from five individual protein subunits, which include combinations of α1–6, β1–3, γ1–3, δ, ε, π and θ (Pirker et al., 2000). GABAARs consist of a large number of receptor-subtypes based
Results
VAChT labeled varicosities appeared with moderately high density in PnO. When axon fibers were visible, most coursed within the plane of section where they branched frequently and expressed a high number of varicosities. Most of these varicosities resembled beads on a string, which we interpret as boutons en passant. To a lesser extent, boutons terminaux also were observed based on their terminal position along an axon fiber. Some varicosities appeared to be apposed to somata, but the majority did not. Cholinergic varicosities were found in a wide range of sizes from approximately 1 to 5 μm in diameter. Immuno-label for the γ2 subunit had a punctate appearance consistent with being associated with clustered GABAARs, as has been described for the γ2 subunit (Essrich, et al., 1998). The
Fig. 1 – Fluorescence confocal image of a representative field in PnO showing MAP2-labeled dendrites (red) and punctate label for the γ2 GABAAR-subunit (blue). The γ2-label is not found inside dendrites (arrows). The MAP2/γ2 label indicated by arrow head is not colocalized in the z-dimension.
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Table 1 – Sampled counts of cholinergic varicsities and γ2 GABAAR-subunit label. Subj.
Total
Total
Total double
% Double
% Double
Rat ♯1 Rat ♯2 Rat ♯3 Rat ♯4 Mean SEM
VAChTþ 263 201 249 154 216.75 721.45
γ 49 46 56 56 51.75 72.19
VAChTþ/γ 16 20 22 12 17.5 71.92
VAChT 6.08 9.95 8.84 7.79 8.17 70.71
γ2 32.65 43.48 39.29 21.43 34.21 74.16
Counts are the totals of fields in PnO on two sections from each rat. % Double VACHT, percentage of total VAChT labeled varicosities colabled for the γ2 subunit. % Double γ2, percentage of total punctate γ2 label found in VAChT labeled varicosities. SEM, standard error of the mean.
Fig. 2 – Fluorescence confocal image of representative double-labeled varicosities in PnO co-expressing VAChT (blue) and the γ2 GABAAR-subunit (red) apposed by varicosities labeled for VGAT (green). (A)–(F) a single field in PnO showing individual, (A)–(C) and combinations of color channels, (D)–(F). (D) VAChT/γ2; and (E), VAChT/VGAT; (F) all three channels merged. Scale bar in F, 5 μm.
on their subunit composition and this gives rise to a multiplicity of functional and pharmacological properties (reviewed in Fritschy and Brünig, 2003 and Olsen and Sieghart, 2009). Individual subunits have been visualized utilizing immunohistochemistry (Fritschy et al., 1992) and most of the subunits tested were observed to be expressed in PnO (Pirker et al., 2000). The presence of a subunit is putative evidence for the expression of functional GABAAR-subtypes containing that subunit. Colocalization of VAChT labeled varicosities and the γ2 GABAAR-subunit in the PnO is consistent with direct presynaptic control over ACh release by GABA. γ2 subunit immunoreactivity has been reported in processes in PnO with moderate density (Pirker et al., 2000). This mechanism is supported further by local blockade of GABAARs in PnO of cat and mouse resulting in increased levels of ACh (Flint et al., 2010; Vazquez and Baghdoyan, 2004), suggesting that control by GABA is inhibitory to ACh-release in PnO. Cholinergic agonists and GABAAR antagonists injected into the PnO of
rat induced increase in REM sleep with very similar characteristics and both drug-inductions were blocked by the muscarinic receptor antagonist atropine (Marks et al., 2008). Thus, the mechanism of GABAAR antagonism is dependent upon the function of local cholinergic receptors. The presence of GABAARs on cholinergic boutons in PnO favors the direct action of GABAAR blockade on these presynaptic terminals to increase ACh release and result in a cholinergic REM sleep induction mediated through muscarinic receptors in PnO. Additional mechanisms of GABA's action on REM sleep cannot be excluded. GABAARs are ubiquitously expressed in the PnO (Pirker et al., 2000). Data presented here support the conclusion that specific GABAARs mediating REM sleep-induction by GABAAR antagonists are located on boutons of cholinergic axon fibers and, at least in part, include a subtype containing the γ2 subunit. The presynaptic mechanism of GABAAR inhibition of AChrelease in PnO is not known. Reported paucity of expression of
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Fig. 3 – Example of a single, double-labeled, cholinergic varicosity in PnO (VAChT) colabeled for the γ2 subunit and clearly apposed by two VGAT labeled varicosities (arrows). Color assignments same as in Fig. 1. (A)–(C), individual color channels and (D) all three channels merged. Scale bar in (D), 5 μm.
the main Cl extrusion system, potassium-chloride cotransporter-2 (KCC2), in neuronal axons favors a depolarizing action at these sites (reviewed in Blaesse et al., 2009). We have observed a lack of KCC2-immunoreactivity in VAChT-labeled varicose fibers in the PnO (Liang and Marks, 2011). GABAAR-mediated depolarization could result in inhibiting ACh-release in PnO through a variety of mechanisms, including inactivation of Naþ channels, Ca2þ channels, and shunting of current (reviewed in Kullmann et al., 2005). These actions would result in reduced exocytotic release of ACh, possibly by blockade of action potential propagation. The lack of expression of γ2-puncta in membranes of somata and dendrites is consistent with γ2 subunit in PnO selectively expressed in axon fibers. This indicates that the major role of GABAARs containing γ2 subunits in PnO is to function in the presynaptic modulation of the release of neurotransmitters. Greater than one-third of the γ2-counts were found in cholinergic boutons consistent with ACh being one neurotransmitter system being modulated by GABA. Which other neurotransmitter systems in PnO express γ2 in their axons and what functions they may have remains to be explored. VAChT-labeled varicosities in PnO, however, were found to colocalize γ2 subunit labeling with only about an 8% incidence. This could occur if γ2 subunit-containing receptor expression were dynamic and drop below the level of detection.
Additionally, multiple GABAAR-subtypes may be distributed among the cholinergic varicosities and not all subtypes contain the γ2 subunit. Alternatively, GABAARs may not be present at every cholinergic bouton and GABA inhibition of ACh release in the PnO may not be acting at each individual presynaptic site of ACh release. One possibility is that cholinergic boutons in PnO containing γ2 subunits are functionally distinct, preferentially providing ACh access to postsynaptic muscarinic receptors specifically involved in REM sleep control. This may include a specific source of cholinergic innervation of PnO or subpopulations from specific sources, for example, the minor proportion of putative mesopontine cholinergic neurons that may provide input to PnO whose activity is selective to REM sleep (Datta and Siwek, 2002; Kayama et al., 1992). Another possibility relates to the nature of the extensive varicose axonal arbors of cholinergic neurons in PnO. These axons could be subjected to GABAmediated depolarization at selective sites along the axon and, through a shunting of current, result in blockade of action potential propagation to distal branches. By this mechanism, an entire region, such as PnO, could effectively be deprived of cholinergic innervation that requires impulse-dependent release. This latter mechanism is consistent with the state-related activity of the majority of mesopontine cholinergic neurons
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discharging at their highest rates in wake and REM sleep (Datta and Siwek, 2002; Kayama et al., 1992). Based on injections of retrograde tracers, the majority of these cholinergic neurons projecting to the pontine reticular formation also have rostral projections including the thalamus (Semba et al., 1990). ACh levels measured by microdialysis in the thalamus are high during both REM sleep and wake compared to NREM sleep (Williams et al., 1994), yet, ACh levels measured in cat pontine reticular formation are the highest in REM sleep alone compared to wake and NREM sleep (Kodama et al., 1990). GABA could act locally through a mechanism blocking impulse-dependent release of ACh in the PnO selectively during wakefulness. Natural reduction of this GABA inhibition during REM sleep, or local pharmacological antagonism of GABAARs, would restore a relationship between activity of cholinergic neurons and ACh release. We suggest it is the increase in ACh release during wakefulness produced by GABAAR antagonists in PnO that results in induction of REM sleep. We identified γ2 as a subunit of at least one GABAAR subtype localized to cholinergic boutons in the PnO. Other subunits constituting these receptors are yet unknown. Nonetheless, just knowledge of γ2 as a subunit permits certain inferences. γ2 containing subtypes are associated with receptors possessing benzodiazepine (BZ) binding sites (Kucken et al., 2000). Additional support for the role of BZ binding sites is our recent finding of induction of REM sleep with the local application of a BZ site inverse agonist in PnO that is also blocked by atropine (Nguyen et al., 2013). Conversely, agonists at the BZ site potentiate the action of GABA and commonly result in reduced REM sleep when prescribed as hypnotics (Lancel, 1999). This is consistent with action of these drugs at receptors in PnO in which antagonizing GABA function increases REM sleep and potentiating function reduces REM sleep (Flint et al., 2010; Sanford, et al., 2003; Vanini and Baghdoyan, 2013). The γ2 subunit is thought to be involved in the mechanism of targeting some receptor subtypes to synaptic locations (Essrich et al., 1998). We have observed double-labeled VAChT/γ2 varicosities in close apposition to varicosities labeled with VGAT (Figs. 2 and 3) and GAD (not shown). This observation is consistent with the GABAARs on cholinergic boutons in rat PnO being postsynaptic to a specific GABAergic innervation. Identifying the source, or sources, of this GABAergic innervation presynaptic to cholinergic terminals in PnO should reveal a critical, up-stream mechanism of REM sleep control. GABAergic neurotransmission in the PnO is likely to constitute a natural mechanism of REM sleep control. GABA
levels, studied with in vivo microdialysis in the pontine reticular formation of the cat, were found to undergo significant alterations with spontaneous changes in the states of arousal (Vanini et al., 2011). Levels of GABA were significantly lower during natural REM sleep compared to both NREM sleep and wakefulness. This is the reciprocal of the relationship found for ACh, which is significantly higher during REM sleep compared to both NREM sleep and wakefulness (Kodama et al., 1990). This reciprocal relationship is consistent with GABAergic inhibition of ACh release in PnO and the central role both neurotransmitters play in control of REM sleep.
4.
Conclusion
GABAARs are expressed in cholinergic varicose axon fibers within the pontine reticular formation's REM sleep induction zone supporting the direct action of GABA on cholinergic boutons. Synaptic release of GABA during wakefulness would inhibit ACh release while reduced GABA-action would result in increased ACh release promoting REM sleep.
5.
Experimental procedure
All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the local Institutional Animal Care and Use Committee at the Dallas VA. All efforts were made to minimize the number of animals used and their suffering.
5.1.
Tissue collection
Four adult male, Long-Evans Hooded rats (Harlan, Indianapolis, IN, USA) weighing between 180 and 250 g were housed under standard conditions on a 12/12 h light/dark schedule. At between 3 and 5 h after lights-on, rats were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and then perfused through the ascending aorta with 30 ml heparinized saline and 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 30 min. The brains were removed, blocked in the coronal plane, post-fixed in the same fixative for 1 h and cryoprotected by immersion in 20% sucrose in PB. Serial, coronal sections, 20 μm-thick were cut on a freezing microtome (SM2000R, Leica, Nussloch, Germany) through the pons, and collected in 0.1 M PB, with 0.005% sodium azide. The region of interest was the caudal aspect of the PnO, based on the high degree of effectiveness of
Table 2 – Primary antibodies used in the study. Antigen
Species raised in
Source and code
Dilution
Specificity reference
VAChT
Goat
Chemicon, AB1578 Temecula, CA, USA
1:2000
γ2
Rabbit
PhosphoSolutions 832-GG2C Auroray, CO, USA
1:500
VGAT GAD67
Mouse Mouse
Synaptic Systems 131 011 Göttingen, Germanyf Chemicon/Millipore MAB5406 Temecula, CA, USA
1:1000 1:1000
MAP2
Chicken
Novus, NB300–213 Littleton, CO, USA
1:10,000
Arvidsson et al. (1997) Henny and Jones (2006) Sperk et al. (1997) Pirker et al. (2000) Takamori et al. (2000) Verea et al. (2005) Watanabe et al. (2005) Li et al. (2010)
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GABAAR antagonists to induce REM sleep at this site (Marks et al., 2008). This was defined as the area of the pontine reticular formation included in the 0.5 mm caudal to the decussation of the superior cerebellar peduncle, approximately 8.3–8.8 mm posterior to the bregma suture (Paxinos and Watson, 1997).
5.2. Immunohistochemistry, microscopy and image acquisition Free-floating sections were primarily blocked with 3% normal donkey serum and 0.3% Triton X-100 in PBS for 60 min and then incubated in a mixture of primary antibodies for VAChT and γ2 or MAP2 and γ2 (double staining), or VAChT, γ2 and, GABAergic neuronal markers, vesicular GABA transporter (VGAT) or glutamic acid decarboxylase-67 (GAD67;triple staining) for 18–20 h at 4 1C (see Table 2). After washing with PBS, sections were incubated with fluorescent-tagged (Cy2, Cy3 and Cy5) secondary antibodies for 3 h (Jackson Immuno Research Labs, West Grove, PA, USA). After 30 min wash with PBS, sections were mounted and cover-slipped with mounting medium containing glycerin and phenylenediamine. Negative controls for antibody labeling indicated a lack of specific staining when primary or secondary antibodies were omitted. Confocal microscopy was performed to examine the relationship between VAChT and γ2 positive varicosities as well as the relationships among VAChT, γ2 and, VGAT or GAD67 positive varicosities in PnO. The relationship of γ2 to dendrites was assessed using MAP2 labeling (De Camilli et al., 1984). Simultaneous epifluorescence (Cy2 and Cy3, or Cy2, Cy3 and Cy5) images were obtained using a laser-scanning confocal microscope (LSM510-META, Zeiss, Göttingen, Germany) with a 100 objective (oil; N.A., 1.4; pin hole, 1 Airy Unit all channels). Excitation was provided by three lasers with excitation wave lengths of 488, 543, and 633 nm. Laser-scanning sectioning in the Z plane was performed using multitrack scanning. Two coronal sections, separated by 250 μm, were randomly chosen from each of the four rats. Starting 2 μm below the surface of each section, a z-stack of at least 6 optical slices was obtained in 0.33 μm steps. While viewing fluorescence emission solely from the VAChT-label, an xy-field was chosen around the center of the PnO to insure a sufficient number of cholinergic varicosities for counting. Colocalization was assessed by viewing all immuno-labels in the field on adjacent Z sections. No significant differences in counts were found between the pairs of sections and they were combined for total VAChT, γ2 and their colocalization. Additional sections were chosen to determine the relationship of γ2 with MAP2 labeling. Confocal images were imported into Photoshop software (CS4, Adobe, San Jose, CA, USA) for composition of merged images and global adjustments of brightness and contrast. Some confocal images were imported into Volocity software (v. 5.2, Improvision, Waltham, MA, USA) for deconvolution and 3-D reconstruction.
Acknowledgments This work was supported by Department of Veterans Affairs Merit Review. We thank Ginger (Jinghua) Zhou for her expert assistance in the histological preparation of brain tissue.
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Ahnaou, A., Basille, M., Gonzalez, B., Vaudry, H., Hamon, M., Adrien, J., Bourgin, P., 1999. Long-term enhancement of REM sleep by the pituitary adenylyl cyclase-activating polypeptide (PACAP) in the pontine reticular formation of the rat. Eur. J. Neurosci. 11, 4051–4058. Ahnaou, A., Laporte, A.M., Ballet, S., Escourrou, P., Hamon, M., Adrien, J., Bourgin, P., 2000. Muscarinic and PACAP receptor interactions at pontine level in the rat: significance for REM sleep regulation. Eur. J. Neurosci. 12, 4496–4504. Arvidsson, U., Riedl, M., Elda, R., Meister, B., 1997. Vesicular acetylcholine transporter (VAChT) protein: a novel and unique marker for cholinergic neurons in the central and peripheral nervous system. J. Comp. Neurol. 378, 454–467. Blaesse, P., Airaksinen, M.S., Rivera, C., Kaila, K., 2009. Cationchloride cotransporters and neuronal function. Neuron 61, 820–838. Bourgin, P., Ahnaou, A., Laporte, A.M., Hamon, M., Adrien, J., 1999. Rapid eye movement sleep induction by vasoactive intestinal peptide infused into the oral pontine tegmentum of the rat may involve muscarinic receptors. Neuroscience 89, 291–302. Bourgin, P., Escourrou, P., Gaultier, C., Adrien, J., 1995. Induction of rapid eye movement sleep by carbachol infusion into the pontine reticular formation in the rat. NeuroReport 6, 532–536. Bourgin, P., Lebrand, C., Escourrou, P., Gaultier, C., Franc, B., Hamon, M., Adrien, J., 1997. Vasoactive intestinal polypeptide microinjections into the oral pontine tegmentum enhance rapid eye movement sleep in the rat. Neuroscience 77, 351–360. Datta, S., Siwek, D.F., 2002. Single cell activity patterns of pedunculopontine tegmentum neurons across the sleep–wake cycle in the freely moving rats. J. Neurosci. Res. 70, 611–621. De Camilli, P., Miller, P.E., Navone, F., Theurkauf, W.E., Vallee, R.B., 1984. Distribution of microtubule-associated protein 2 in the nervous system of the rat studied by immunofluorescence. Neuroscience 11, 819–846. Essrich, C., Lorez, M., Benson, J.A., Fritschy, J.-M., Lüscher, B., 1998. Postsynaptic clustering of GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. Nat. Neurosci. 1, 563–571. Flint, R.R., Chang, T., Lydic, R., Baghdoyan, H.A., 2010. GABAA receptors in the pontine reticular formation of C57BL6J mouse modulate neurochemical, electrographic, and behavioral phenotypes of wakefulness. J. Neurosci. 30, 12301–12309. Fritschy, J.-M., Benke, D., Mertens, S., Oertel, W.H., Bachi, T., Möhler, H., 1992. Five subtypes of type A γ-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies. Proc. Natl. Acad. Sci. USA 89, 6726–6730. Fritschy, J.-M., Brünig, I., 2003. Formation and plasticity of GABAergic synapses: physiological mechanisms and pathophysiological implications. Pharmacol. Ther. 98, 299–323. Gilmor, M.L., Nash, N.R., Roghani, A., Edwards, R.H., Yi, H., Hersch, S.M., Levey, A.I., 1996. Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles. J. Neurosci. 16, 2179–2190. Henny, P., Jones, B.E., 2006. Vesicular glutamate (VgluT), GABA (VGAT), and acetylcholine (VAChT) transporters in basal forebrain axon terminals innervating the lateral hypothalamus. J. Comp. Neurol. 496, 53–467. Kayama, Y., Ohta, M., Jodo, E., 1992. Firing of ‘possibly’ cholinergic neurons in the rat laterodorsal tegmental nucleus during sleep and wakefulness. Brain Res. 569, 210–220.
64
brain research 1543 (2014) 58–64
Kodama, T., Takahashi, Y., Honda, Y., 1990. Enhancement of acetylcholine release during paradoxical sleep in the dorsal tegmental field of the cat brain stem. Neurosci. Lett 114, 277–282. Kucken, A.M., Wagner, D.A., Ward, P.R., Teissére, J.A., Boileau, A.J., Czajkowski, C., 2000. Identification of benzodiazepine binding site residues in the γ2 subunit of the γ-aminobutyric acidA receptor. Mol. Pharmacol. 57, 932–939. Kullmann, D.M., Ruiz, A., Rusakov, D.M., Scott, R., Semyanov, A., Walker, M.C., 2005. Presynaptic, extrasynaptic and axonal GABAA receptors in the CNS: where and why?. Prog. Biophys. Mol. Biol. 87, 33–46. Lancel, M., 1999. Role of GABAA receptors in the regulation of sleep: initial sleep responses to peripherally administered modulators and agonists. Sleep 22, 33–42. Li, Y., Serwanski, D.R., Miralles, C.P., Fiondella, C.G., LoTurco, J.J., Rubio, M.E., De Blas, A.L., 2010. Synaptic and non-synaptic localization of protocadherin-γC5 in the rat brain. J. Comp. Neurol. 518, 3439–3463. Liang C.-L., Marks, G.A., 2011. On expression of certain proteins in mesopontine cholinergic neurons at their soma and terminals in the pontine reticular formation. Program ♯505.12, Neuroscience Meeting Planner, Society for Neuroscience, Washington, DC, Online. MacDermott, A.B., Role, L.W., Siegelbaum, S.A., 1999. Presynaptic ionotropic receptors and the control of transmitter release. Annu. Rev. Neurosci. 22, 443–485. Marks, G.A., Birabil, C.G., 1998. Enhancement of rapid eye movement sleep in the rat by cholinergic and adenosinergic agonists infused into the pontine reticular formation. Neuroscience 86, 29–37. Marks, G.A., Sachs, O.W., Birabil, C.G., 2008. Blockade of GABA, type A, receptors in the rat pontine reticular formation induces rapid eye movement sleep that is dependent upon the cholinergic system. Neuroscience 156, 1–10. Marks, G.A., Shaffery, J.P., Speciale, S.G., Birabil, C.G., 2003. Enhancement of rapid eye movement sleep in the rat by actions at A1 and A2a adenosine receptor subtypes with a differential sensitivity to atropine. Neuroscience 116, 913–920. Nguyen, T.Q., Liang, C.-L., Marks, G.A., 2013. GABAA receptors implicated in REM sleep control express a benzodiazepine binding site. Brain Res. 1527, 131–140. Olsen, R.W., Sieghart, W., 2009. GABA A receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology 60, 243–260.
Paxinos, G., Watson, C., 1997. The Rat Brain in Stereotaxic Coordinates: Compact, 3rd Ed. Academic Press, San Diego (CD-ROM). Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W., Serk, G., 2000. GABAA receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101, 815–850. Sanford, L.D., Tang, X., Xiao, J., Ross, R.J., Morrison, A.R., 2003. GABAergic regulation of REM sleep in reticularis pontis oralis and caudalis in rats. J. Neurophysiol. 90, 938–945. Semba, K., Reiner, P.B., Fibiger, H.C., 1990. Single cholinergic mesopontine tegmental neurons project to both the pontine reticular formation and the thalamus in the rat. Neuroscience 38, 643–654. Sperk, G., Schwarzer, C., Tsunashima, K., Fuchs, K., Sieghart, W., 1997. GABAA receptor subunits in the rat hippocampus I: immunocytochemical distribution of 13 subunits. Neuroscience 80, 987–1000. Takamori, S., Riedel, D., Jahn, R., 2000. Immunoisolation of GABAspecific synaptic vesicles defines a functionally distinct subset of synaptic vesicles. J. Neurosci. 20, 4904–4911. Vanini, G., Wathen, B.L., Lydic, R., Baghdoyan, H.A., 2011. Endogenous GABA levels in the pontine reticular formation are greater during wakefulness than during rapid eye movement sleep. J. Neurosci. 31, 2649–2656. Vanini, G., Baghdoyan, H.A., 2013. Extrasynaptic GABAA receptors in rat pontine reticular formation increase wakefulness. Sleep 36, 337–343. Vazquez, J., Baghdoyan, H.A., 2004. GABAA receptors inhibit acetylcholine release in cat pontine reticular formation: implications for REM sleep regulation. J. Neurophysiol. 92, 2198–2206. Verea, E., Nácher, J., Blasco-Ibáñez, J.M., Gómez-Climen, M.A., Castillo-Gómez, E., Crespo, C., Martinez-Guijarro, F.J., 2005. PSA-NCAM expression in the rat medial prefrontal cortex. Neuroscience 136, 435–443. Watanabe, K., Kamiya, D., Nishiyama, A., Katayama, T., Nozaki, S., Kawasaki, H., Watanabe, Y., Mizuseki, K., Sasai, Y., 2005. Directed differentiation of telencephalic precursors from embryonic stem cells. Nature Neurosci. 8, 288–296. Williams, J.A., Comisarow, J., Day, J., Fibiger, H.C., Reiner, P.B., 1994. State-dependent release of acetylcholine in rat thalamus measured by in vivo microdialysis. J. Neurosci. 14, 5236–5242.