Opioid modulation of GABA release in the rat inferior colliculus

Background 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. Results 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. Conclusion 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.


Background
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][8][9][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 [12]. Recently a fourth related receptor ORL1 activated by the peptide nociceptin has been identified and its distinct pharmacology has been described [13]. 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 [14]. For example, in the periaqueductal gray stimulation of opiate receptors and their associated G-proteins results in the activation of potassium channels [15] while in the hippocampus, inhibition of the GABAergic activity by opioid is independent of potassium channel activation [16].
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 [ 3 H]GABA from inferior colliculus slices

KCl-induced [ 3 H]GABA release
Inferior colliculus slices pre-incubated with [ 3 H]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.
Elution profile of KCl-stimulated [ 3 H]GABA release from inferior colliculus slices Figure 1 Elution profile of KCl-stimulated [ 3 H]GABA release from inferior colliculus slices. Inferior colliculus slices were incubated with 11 nM [ 3 H]GABA for 30 min and perfused (0.5 ml/min) with Krebs buffer for 30 min in a Brandel superfiltration apparatus. Fractions (1 ml) were then collected every 2 min. Krebs buffer containing 25 mM KCl was loaded into the system at times corresponding to fraction 4 and 12 (short black lines) which elicited [ 3 H]GABA release peaks in fractions 6-7 and fraction 14-15. Krebs buffer containing modulating drugs (1 µM morphine-long blue line) was added from the time corresponding to fraction 8. Scintillation fluid (3 ml) was added to the each fractionated eluate and to the solubilized tissue samples and counted. Data are expressed as fractional release which is calculated as the radioactivity released in one fraction divided by the amount of radioactivity present in the tissue just before that fraction. The two peaks from each elution profile are referred to as S1 and S2. The data from a representative experiment are shown.

Morphine modulation of KCl induced [ 3 H]GABArelease
The effect of different concentrations of morphine on KClinduced [ 3 H]GABA release is shown in Figure 2. Both 1 µM and 5 µM but not 100 nM morphine caused a significant decrease of [ 3 H]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 [ 3 H]GABA release. These data strongly indicate that morphine modulates the release of [ 3 H]GABA via activation of opiate receptors. The reduction in [ 3 H]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 [7]. In addition a functional inter-relationship is established between the two systems which could be of physiological significance. Effect of morphine on KCl-stimulted [ 3 H]GABA release. The ratio of the integration of the peaks S2 over S1 was calculated for each elution profile. Morphine (0.1, 1 and 5 µM) and naloxone (10 µM) alone or together were perfused as described in figure 1 but naloxone perfusion was started one fraction earlier. Data represent averages ± SEM. Labels on the x-axis indicate concentration of drugs present during S2. Statistical difference was calculated using one-way ANOVA. The data indicates that 1 and 5 µM morphine caused a statistically significant 16% reduction of [ 3 H]GABA release as compared to control (a) (p < 0.01). This effect of morphine was blocked by the antagonist naloxone (c) which had no effect on its own (b). 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 [ 3 H]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 [ 3 H]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 µopioid 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 fluoro-chromes 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.

Conclusions
This study has demonstrated that in the rat inferior collic-  Figure 2 but there was no statistical difference between experiments with or without BIM (One way ANOVA). Confocal microscopy of cryosection of inferior colliculus slices stained with anti glutamic acid decarboxylase (green) and anti µ-opioid receptor antibodies (red) Figure 5 Confocal microscopy of cryosection of inferior colliculus slices stained with anti glutamic acid decarboxylase (green) and anti µ-opioid receptor antibodies (red). A : Central nucleus of inferior colliculus. B: Pericentral nucleus of inferior colliculus. Arrow head: example of GABAergic neurons not containing µ-opioid receptor (Green). Arrows: example of GABAergic neuron containing µ-opioid receptor (Yellow).
A B 20µ µ µ µm 20µ µ µ µm 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.

Slices preparations
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.

Neurotransmitter release
As previously described, slices were incubated in 5 ml oxygenated (95% 0 2 / 5% CO 2 ) Krebs buffer containing GABA transaminase inhibitor aminooxyacetic acid (100 µM) at 32°C for 5 min [26].  (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 conju-gated 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).