Opioid modulation of GABA release in the rat inferior colliculus
BMC Neuroscience volume 5, Article number: 31 (2004)
The inferior colliculus, which receives almost all ascending and descending auditory signals, plays a crucial role in the processing of auditory information. While the majority of the recorded activities in the inferior colliculus are attributed to GABAergic and glutamatergic signalling, other neurotransmitter systems are expressed in this brain area including opiate peptides and their receptors which may play a modulatory role in neuronal communication.
Using a perfusion protocol we demonstrate that morphine can inhibit KCl-induced release of [3H]GABA from rat inferior colliculus slices. DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin) but not DADLE ([D-Ala2, D-Leu5]-enkephalin or U69593 has the same effect as morphine indicating that μ rather than δ or κ opioid receptors mediate this action. [3H]GABA release was diminished by 16%, and this was not altered by the protein kinase C inhibitor bisindolylmaleimide I. Immunostaining of inferior colliculus cryosections shows extensive staining for glutamic acid decarboxylase, more limited staining for μ opiate receptors and relatively few neurons co-stained for both proteins.
The results suggest that μ-opioid receptor ligands can modify neurotransmitter release in a sub population of GABAergic neurons of the inferior colliculus. This could have important physiological implications in the processing of hearing information and/or other functions attributed to the inferior colliculus such as audiogenic seizures and aversive behaviour.
Sounds are first converted into neuronal signals in the inner ear and then conveyed to the cerebral cortex via a number of discrete brain areas including the inferior colliculus. Each of these areas receives ascending pathways carrying signals from one or both ears and descending pathways from higher brain centres. The current knowledge of the neurochemical events occurring at each of these brain centres is limited [1, 2]. In the inferior colliculus studies have been carried out to characterise the role of GABAergic neurons especially in sound localisation which is believed to be one of the main functions of this brain area [3, 4]. Additionally the inferior colliculus has ben implicated in audiogenic seizures and aversive behaviour in which GABAergic neurons may also play an important role. [5, 6]
The neuronal communication occurring in the inferior colliculus is likely to be influenced by modulatory systems such as those of peptidergic neurotransmitters. Opiate receptor gene expression, immunoreactivity and activity in the inferior colliculus have been described [7–10] although detailed studies on the effect of opiate on GABA neurotransmitter release in this brain regions have not been carried out.
Three classes of opiate peptides endorphins, dynorphins and enkephalins activate μ, κ and δ-opiate receptors subtypes respectively . Recently a fourth related receptor ORL1 activated by the peptide nociceptin has been identified and its distinct pharmacology has been described . All opiate receptors are associated with either Go or Gi subunits and they mediate inhibitory actions including pre-synaptic inhibition of neurotransmitter release. Different mechanisms of inhibition of neurotransmitter release have been reported in various tissues and neurons . For example, in the periaqueductal gray stimulation of opiate receptors and their associated G-proteins results in the activation of potassium channels  while in the hippocampus, inhibition of the GABAergic activity by opioid is independent of potassium channel activation .
In previous work we have established the presence and distribution of opiate receptors in the adult and developing rat cochlea suggesting that the opiate system has a role in hearing function [17, 18]. In order to extend our knowledge of the role of opiate system in hearing it is necessary to characterise its presence and role also in the auditory pathways. Our hypothesis was that opiate peptides can modulate synaptic function in the auditory pathways by pre-synaptically altering the release of other neurotransmitters. To test this hypothesis we have used opiate drugs to inhibit the release of [3H]GABA from inferior colliculus slices
Results and Discussion
KCl-induced [3H]GABA release
Inferior colliculus slices pre-incubated with [3H]GABA were perfused for 30 min and stimulated twice with 25 mM KCl to elicit neurotransmitter release. The eluate was collected in 1 ml fractions and the released radioactivity was assessed by scintillation counting. Figure 1 shows two examples of typical release profiles from slices perfused with either Krebs buffer throughout (control) or with Krebs buffer for fractions 1–7 and with Krebs containing 1 μM morphine for the remaining fractions, where both samples were stimulated with KCl at the time corresponding to fraction 4 and 12. The two peaks were referred to as S1 and S2 and occured approximately 2 fractions after the application of KCl due to the buffer volume contained in the tubes feeding into the incubation chamber. Values of the radioactivity eluted are expressed as fractional release which is the ratio of the radioactivity released in a particular fraction divided by the total amount of radioactivity contained in the tissue immediately prior to that fraction. The variation in the value of S1 of the two profiles shown in Fig 1, both induced by KCl alone, reflects the variation in amount of tissue present in each of the elution chambers and illustrates the need for utilising the ratio of the two peaks (S2/S1) of each elution profile as a mean to detect the effect of the modulating drug.
Morphine modulation of KCl induced [3H]GABArelease
The effect of different concentrations of morphine on KCl-induced [3H]GABA release is shown in Figure 2. Both 1 μM and 5 μM but not 100 nM morphine caused a significant decrease of [3H]GABA release from the inferior colliculus slices. The effect of 1 μM morphine was antagonised by the antagonist naloxone (10 μM) which was perfused from one fraction before the addition of morphine. The perfusion of naloxone alone did not cause a significant effect on [3H]GABA release. These data strongly indicate that morphine modulates the release of [3H]GABA via activation of opiate receptors. The reduction in [3H]GABA release calculated as the change in S2/S1 ratios in the presence and absence of morphine during S2 was 16% (p < 0.01). These data agree with previous reports on the presence of both GABA neurons and opiate receptors and peptides in the inferior colliculus . In addition a functional inter-relationship is established between the two systems which could be of physiological significance.
Specific role of μ opiate receptors
In order to establish which of the opiate receptor subtypes are involved in the modulation of the [3H]GABA release, morphine was substituted by either 1 μM DAMGO, DADLE or U69593 which specifically activate μ, δ and κ opiate receptors respectively (Fig. 3). Only DAMGO (1 μM) had a significant effect on [3H]GABA release, an effect that was again antagonised by naloxone. DAMGO, as well as morphine, reduced the amount of [3H]GABA release by 16% (p < 0.01) indicating that only μ opioid receptors participate in the regulation of GABA release. Higher concentrations of DAMGO (5 μM) did not have greater effects on [3H]GABA release (not shown). Data from our lab (unpublished) and from others [9, 10] indicate that mRNA transcripts or receptor binding for all three opiate receptor subtypes are present in the inferior colliculus. Further work is required to establish the roles of the δ - and κ-opioid receptors in the inferior colliculus.
A possible explanation for the relatively low effect of opiate agonist on [3H]GABA release (16%) could be that during the exposure to opiate agonists, down-regulation of the opiate receptors may occur . To address this possibility experiments were carried out in the presence of the protein kinase C inhibitor bisindolylmaleimide I (BIM). BIM has been shown to inhibit receptor desensitisation [20, 21] and to reverse tolerance to opiate drugs which involves opiate receptor desensitisation. [22, 23]. Because BIM is solubilised in DMSO additional control assays were carried out to check for the effect of the solvent. The results indicate (Fig. 4) that BIM had no effect on the extent of morphine inhibition of [3H]GABA release. While there is no direct proof that BIM had its reported effect on the tissue, the data indicate that receptor desensitisation may not be the cause of the relatively low percentage effect of morphine.
Co-localisation of μ-opiate receptors and GABAergic neurons
Another possible explanation for the small (16%) reduction of [3H]GABA release by opiate agonists may be the limited number of GABAergic neurons that express opiate receptors. To address this question inferior colliculi slices were double labelled with guinea pig antibodies against μ-opioid receptors and with rabbit antibodies against glutamic acid decarboxylase (the enzyme uniquely responsible for the synthesis of GABA) Species specific secondary antibodies conjugated to red and green fluorochromes allowed the detection of both antigens on the same slide (Fig. 5). Although these results were qualitative it was evident that staining of glutamic acid decarboxylase was more extensive than that of μ-opiate receptors and that only a few GABAergic neurons showed co-localisation of μ-opiate receptors. These data are consistent with the proposal that only a sub-population of GABAergic neurons are under the influence of opiate receptors. Establishing the nature of the GABAergic neurons that express opiate receptors will be an important task in understanding the role of opiate signalling in the inferior colliculus.
This study has demonstrated that in the rat inferior colliculus slices opiate agonists can inhibit KCl-induced [3H]GABA release via activation of the μ-opiate receptor subtype. The amount of [3H]GABA released in presence of opiate agonists was 16% lower than in control slices. This relatively low level of decrease is probably not due to receptor desensitization occurring during the assay but rather to a relatively small population of GABAergic neurons in the inferior colliculus expressing μ-opiate receptors. The small effect of the opiate compounds could also indicate that modulation of GABA release is not their major role, but it could still be of physiological significance.
Together with its reported role in audiogenic seizures and aversive behaviour, the inferior colliculus is an important neuronal centre for auditory processing containing both ascending and descending fibres. The identification of the role of opiate peptides and possibly other modulatory system in the inferior colliculus and other areas of the auditory pathway may allow a better understanding of the mechanism of the hearing system and possibly offer a target for therapeutic intervention in hearing dysfunction. Alternatively, elucidation of the role opiate peptides in the inferior colliculus could provide information about regulation of audiogenic seizures and aversive behaviour.
Opiate agonist and antagonists, (Morphine, DAMGO [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin, DADLE [D-Ala2, D-Leu5]-enkephalin, U69593 and naloxone were purchased from SIGMA, UK. Antibodies against μ-opiate receptor AB1774 (guinea pig polyclonal) and glutamic acid decarboxylase AB1511 (rabbit polyclonal) and species specific pre-absorbed secondary antibodies (donkey anti rabbit IgG FITC and donkey anti guinea pig IgG rhodamine) were purchased from Chemicon UK. Both antibodies were raised against synthetic peptides, and have been used in several immunocytochemical investigations of rat tissue. [24, 25] AB1511 recognises the two isoforms of the enzyme in a Western blot (65/68 KDa) while antibody AB1511 recognises μ-opiate receptors immunocytochemically in the same tissues as other simlilar antibodies and by insitu hybridisation (Chemicaon data sheets, http://www.chemicon.com) Krebs carbonate buffer: NaCl 118 mM, KCl 4.84 mM, CaCl2 2.4 mM, NaHCO3 25 mM, MgSO4 1.8 mM KH2PO4 1.2 glucose 9.5 mM.
Sprague Dawley rats, approximately 200 g, were obtained from UCL Biological Services. All animal experiments were carried out in accordance to the Animal (Scientific Procedure) Act 1986, UK.
Rats were stunned and killed by cervical dislocation. The skull was opened and the whole brain removed. The inferior colliculus was dissected out by two coronal transections, the first between the cerebellum and the inferior colliculus and the second between the inferior colliculus and the superior coliculus. The slice was placed horizontally and medullar tissue ventral to the inferior colliculus was removed. The inferior colliculus was then placed on a tissue chopper and sliced into 250 μm coronal sections. Individual slices were separated under a dissecting microscope in Krebs buffer.
As previously described, slices were incubated in 5 ml oxygenated (95% 02 / 5% CO2) Krebs buffer containing GABA transaminase inhibitor aminooxyacetic acid (100 μM) at 32°C for 5 min . [3H]GABA was added to give a final concentration of 11 nM and incubated in a shaking water bath for 30 min. Slices were distributed into 6 superfusion chambers between filter papers (Brandel Superfusion System) and perfused at 0.5 ml/min with oxygenated Krebs buffer. Following a 30 min perfusion required to reach a steady state (non-stimulated) [3H]GABA release, 2 min fractions (1 ml) were collected. In order to evoke sub-maximal GABA release the slices were perfused for 2 min with 25 mMKCl at 6–8 min and 22–24 min of the fractionation time (fraction 4 and 12). At the end of each experiment (17 fractions) the tissue and the filter papers were collected and incubated with 500 μl Soluene-350 for 20 min and neutralised with 200 μl of glacial acetic acid. Scintillant (Packard, Ultima Gold, 3 ml) was added to all tissue samples and eluted fractions and the radioactivity was measured by scintillation counting (Wallac 1409). Each drug treament was repeated in several experiments as indicated in the figures and the ratios of the S2/S1 peaks were averaged. Statistical significance of the effect of treatments was analysed by one way ANOVA using Excel (Microsoft, USA)
Inferior colliculi were dissected as decribed above and fixed in 4% paraformaldehyde in phosphate buffer saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4.7H2O, 1.4 mM KH2PO4 pH 7.3) for 1 hour, washed 3 times in PBS and incubated overnight at 4°C in 30%sucrose in PBS. Coronal sections (20 μm) were cut with a cryostat and collected on poly-lysine coated glass slides and allowed to dry. The sections were blocked in 10% normal donkey serum diluted in PBS containing 0.25% bovine serum albumin and 0.1% Triton X-100 (PBS-A) for 30 min at room temperature. Subsequently, they were incubated for 12 hours at 4°C with the combination of primary antibodies (rabbit anti GAD and guinea pig anti mu opioid receptor) diluted 1:500 in PBS containing 1% bovine serum albumin and 0.3% Triton X-100 (PBS-B).. Slides were washed 3 × 10 min with PBS-B and incubated with secondary antibodies diluted 1:200 in PBS-B, for 2 hours at room temperature. The secondary antibodies used were donkey anti rabbit conjugated with fluorescein (AP182F) and donkey anti guinea pig conjugated with rhodamine (AP182R). Finally, the sections were rinsed in PBS-B for 10 min and in PBS for 2 × 10 min and then mounted in Citifluor (Agar). The immunoreactivity was visualized under the confocal microscopy (LSM 510 META Carl Zeiss, Germany).
Jones EG: Chemically defined parallel pathways in the monkey auditory system. Ann N Y Acad Sci. 2003, 999: 218-33. 10.1196/annals.1284.033.
Musiek FE, Hoffman DW: An introduction to the functional neurochemistry of the auditory system. Ear Hear. 1990, 11: 395-402.
McAlpine D, Palmer AR: Blocking GABAergic inhibition increases sensitivity to sound motion cues in the inferior colliculus. J Neurosci. 2002, 22: 1443-1453.
Pollak GD, Burger RM, Park TJ, Klug A, Bauer EE: Roles of inhibition for transforming binaural properties in the brainstem auditory system. Hear Res. 2002, 168: 60-78. 10.1016/S0378-5955(02)00362-3.
Garcia-Cairasco N: A critical review on the participation of inferior colliculus in acoustic-motor and acoustic-limbic networks involved in the expression of acute and kindled audiogenic seizures. Hear Res. 2002, 168: 208-22. 10.1016/S0378-5955(02)00371-4.
Troncoso AC, Osaki MY, Mason S, Borelli KG, Brandao ML: Apomorphine enhances conditioned responses induced by aversive stimulation of the inferior colliculus. Neuropsychopharmacology. 2003, 28: 284-91. 10.1038/sj.npp.1300034.
Kalyuzhny AE, Dooyema J, Wessendorf MW: Opioid-and GABA(A)-receptors are co-expressed by neurons in rat brain. Neuroreport. 2000, 11: 2625-2628.
Mackay KB, McCulloch J: Distribution of effects of the kappa-opioid agonist CI-977 on cerebral glucose utilization in rat brain. Brain Res. 1994, 642: 160-168. 10.1016/0006-8993(94)90918-0.
DePaoli AM, Hurley KM, Yasada K, Reisine T, Bell G: Distribution of kappa opioid receptor mRNA in adult mouse brain: an in situ hybridization histochemistry study. Mol Cell Neurosci. 1994, 5: 327-335. 10.1006/mcne.1994.1039.
Gouarderes C, Tellez S, Tafani JA, Zajac JM: Quantitative autoradiographic mapping of delta-opioid receptors in the rat central nervous system using [125I][D.Ala2]deltorphin-I. Synapse. 1993, 13: 231-240.
Mansour A, Fox CA, Thompson RC, Akil H, Watson SJ: mu-Opioid receptor mRNA expression in the rat CNS: comparison to mu-receptor binding. Brain Res. 1994, 643: 245-265.
Jordan BA, Cvejic S, Devi LA: Opioids and their complicated receptor complexes. Neuropsychopharmacology. 2000, 23 (4 Suppl): S5-S18. 10.1016/S0893-133X(00)00143-3.
Calo' G, Rizzi A, Bigoni R, Guerrini R, Salvadori S, Regoli D: Pharmacological profile of nociceptin/orphanin FQ receptors. Clin Exp Pharmacol Physiol. 2002, 29: 223-228. 10.1046/j.1440-1681.2002.03633.x.
Miller RJ: Presynaptic receptors. Annu Rev Pharmacol Toxicol. 1998, 38: 201-227. 10.1146/annurev.pharmtox.38.1.201.
Vaughan CW, Ingram SL, Connor MA, Christie MJ: How opioids inhibit GABA-mediated neurotransmission. Nature. 1997, 390: 611-614. 10.1038/37610.
Capogna M, Gahwiler BH, Thompson SM: Mechanism of mu-opioid receptor-mediated presynaptic inhibition in the rat hippocampus in vitro. J Physiol. 1993, 470: 539-558.
Phansuwan-Pujito P, Saleema L, Mukda S, Tongjaroenbuangam W, Jutapakdeegul N, Casalotti SO, Forge A, Dodson H, Govitrapong P: The opioid receptors in inner ear of different stages of postnatal rats. Hear Res. 2003, 184: 1-10. 10.1016/S0378-5955(03)00163-1.
Jongkamonwiwat N, Phansuwan-Pujito P, Sarapoke P, Chetsawang B, Casalotti SO, Forge A, Dodson H, Govitrapong P: The presence of opioid receptors in rat inner ear. Hear Res. 2003, 181: 85-93. 10.1016/S0378-5955(03)00175-8.
Lefkowitz RJ, Caron MG: Ciba-Geigy award for outstanding research. Regulation of adrenergic receptor function by phosphorylation. J Mol Cell Cardiol. 1986, 18: 885-895.
Tang H, Shirai H, Inagami T: Inhibition of protein kinase C prevents rapid desensitization of type 1B angiotensin II receptor. Circ Res. 1995, 77: 239-48.
Mela F, Marti M, Ulazzi L, Vaccari E, Zucchini S, Trapella C, Salvadori S, Beani L, Bianchi C, Morari M: Pharmacological profile of nociceptin/orphanin FQ receptors regulating 5-hydroxytryptamine. Eur J Neurosci. 2004, 19: 1317-1324. 10.1111/j.1460-9568.2004.03220.x.
Smith FL, Javed RR, Elzey MJ, Dewey WL: The expression of a high level of morphine antinociceptive tolerance in mice involves both PKC and PKA. Brain Res. 2003, 985: 78-88. 10.1016/S0006-8993(03)03170-6.
Liu JG, Anand KJ: Protein kinases modulate the cellular adaptations associated with opioid tolerance and dependence. Brain Res Brain Res Rev. 2001, 38: 1-19. 10.1016/S0165-0173(01)00057-1.
Rodriguez JJ, Mackie K, Pickel VM: Ultrastructural localization of the CB1 cannabinoid receptor in mu-opioid receptor patches of the rat Caudate putamen nucleus. J Neurosci. 2001, 21: 823-33.
Muzio L, DiBenedetto B, Stoykova A, Boncinelli E, Gruss P, Mallamaci A: Conversion of cerebral cortex into basal ganglia in Emx2(-/-) Pax6(Sey/Sey) double-mutant mice. Nat Neurosci. 2002, 5: 737-745.
Neal MJ, Cunningham JR, Dent Z: Modulation of extracellular GABA levels in the retina by activation of glial P2X-purinoceptors. Br J Pharmacol. 1998, 124: 317-22.
WT was supported by Royal Golden Jubilee Award from the Thailand Research Fund. The work was supported by a CRIG Wellcome Grant (072145) to PG, AF, PP, SOC.
WT carried out the majority of the experiments, NJ carried out initial experiments and established assay conditions, JC provided expertise in neurotransmitter release studies and participated in the design of the study and analysis of the data, PP provided expertise in the double labelling studies, HD, AF and PG participated in the design of the study and analysis of the data, SOC conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.
About this article
Cite this article
Tongjaroenbungam, W., Jongkamonwiwat, N., Cunningham, J. et al. Opioid modulation of GABA release in the rat inferior colliculus. BMC Neurosci 5, 31 (2004). https://doi.org/10.1186/1471-2202-5-31