- Research article
- Open Access
Opioid modulation of GABA release in the rat inferior colliculus
© Tongjaroenbungam et al; licensee BioMed Central Ltd. 2004
- Received: 14 May 2004
- Accepted: 07 September 2004
- Published: 07 September 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.
- Inferior Colliculus
- GABAergic Neuron
- Glutamic Acid Decarboxylase
- Gaba Release
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
KCl-induced [3H]GABA release
Morphine modulation of KCl induced [3H]GABArelease
Specific role of μ opiate receptors
Co-localisation of μ-opiate receptors and GABAergic neurons
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).
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.
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