Muscarinic receptor subtypes in rat dorsal cochlear nucleus

doi:10.1016/0378-5955(95)00131-6

Hearing Research

Volume 89, Issues 1–2, September 1995, Pages 137-145

https://doi.org/10.1016/0378-5955(95)00131-6 Get rights and content

Abstract

We previously reported that responses of spontaneously active rat dorsal cochlear nucleus (DCN) neurons to cholinergic agonists are mediated predominantly by muscarinic receptors. We have now tested the effects of 7 antagonists with differing affinities for the muscarinic receptor subtypes M₁–M₄ on the responses to constant, submaximal doses of carbachol in rat brainstem slices. Each slice was exposed to one or more concentrations of one antagonist applied during extracellular recording of a DCN neuron. The concentrations yielding 50% reduction of test responses (IC₅₀) of regular and bursting neurons were estimated for each antagonist. Correlation coefficients were calculated between log(IC₅₀) values and log(K_i) values of the drugs for the receptor subtypes. Correlation coeffi for both regular and bursting neurons were not significant (P>0.05) for M₁ and M₃, but were significant (P<0.02) for M₄. Bursting but not regular neurons also showed a significant correlation for M₂ (P<0.05). Our results suggest that (1) M₄ contributes to the cholinergic responses in DCN and M₂ may also contribute to the responses of bursting neurons, but the contribution of other subtypes cannot be completely excluded; (2) muscarinic subtypes in DCN probably differ from those reported for cochlea and some brain regions.

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Previous studies have found that tinnitus may be related to spontaneous activity in the dorsal cochlear nucleus[10,11]. Cholinergic receptors have been found in the cochlear nucleus[12,13] indicating that changes in acetylcholine receptors may affect spontaneous activity at this region and therefore affect symptoms such as tinnitus and hyperacusis. Acetylcholine receptor antagonists could block the effects found in the cochlear nucleus that occurs in conjunction with tinnitus and hyperacusis whereby eliminating the perception of these disorders.
The acoustic startle response has been used to evaluate tinnitus and hyperacusis in animal models. Gap induced prepulse inhibition of the acoustic startle reflex (gap-PPI) is affected by tinnitus and loudness changes. Since tinnitus and reduced sound tolerance are commonly seen in elderly, we measured gap-PPI in Fischer 344 rats, an aging related hearing loss model, at different ages: 3-5 months, 9-12 months, and 15-17 months. The startle response was induced by three different intensity of sound: 105, 95 and 85 dB SPL. Gap-PPI was induced by different duration of silent gaps from 1 to 100 ms. When the startle was induced by 105 dB SPL sound intensity, the gap-PPI induced by 50 ms silent gap was significantly lower than those induced by 25 or 100 ms duration, showing a “notch” in the gap-PPI function. The “notch” disappeared with the reduction of startle sound, suggesting the “notch” may be related with hyper-sensitivity to loud sound. As the intensity of the stimulus decreased, the appearance of the hyperacusis-like effect decreased more quickly for the youngest group of rats. We also tested scopolamine, a muscarinic acetylcholine receptor antagonist, and mecamylamine, a nicotinic acetylcholine receptor antagonist, on the effect of gap-PPI. When scopolamine was administered, the results indicated no addition effect on the hyperacusis-like phenomenon in the two older groups. Mecamylamine, the nicotinic antagonist also showed effects on the appearance of hyperacusis on rats in different ages. The information derived from the study will be fundamental for the further research in determining the cause and treatment for hyperacusis.
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The involvement of astrocytes in the cholinergic modulation of the cochlear nucleus has been studied using primary astrocyte cultures prepared from this nucleus. The cells were loaded with the membrane permeable form of the fluorescent Ca²⁺ indicator Fluo-4, and carbachol-induced Ca²⁺ concentration increases were monitored using an imaging system. In the presence of cholinergic stimulation 36.3% of the cells produced Ca²⁺ transients. The time course of the transients was variable; 45.0% of the responding cells showed only a rapid Ca²⁺ concentration increase, while in 50.5% of the astrocytes the fast component was followed by a slow plateau phase. Using muscarine as well as general and more specific cholinergic antagonists (atropine, pirenzepine, 4-DAMP and hexamethonium), the role of the M3 and (to a smaller extent) M1 muscarinic acetylcholine receptors could be demonstrated in the genesis of the carbachol-induced Ca²⁺ transients. The presence of these two subtypes of muscarinic receptors has been confirmed at both mRNA (Q-PCR) and protein (immunocytochemistry) levels. Our data demonstrate the responsiveness of the cochlear astrocytes towards cholinergic stimulation, suggesting that they may have roles in mediating the effects of cholinergic modulation in the rat cochlear nucleus.
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Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the CNS and peripheral nervous system and play an important role in modulating the cell activity and function. We have shown that the cholinergic agonist carbachol reduces the pigeon’s inwardly rectifying potassium channel (pKir2.1) ionic currents in native vestibular hair cells. We have cloned and sequenced pigeon mAChR subtypes M2–M5 and we have studied the expression of all five mAChR subtypes (M1–M5) in the pigeon vestibular end organs (semicircular canal ampullary cristae and utricular maculae), vestibular nerve fibers and the vestibular (Scarpa’s) ganglion using tissue immunohistochemistry (IH), dissociated single cell immunocytochemistry (IC) and Western blotting (WB). We found that vestibular hair cells, nerve fibers and ganglion cells each expressed all five (M1–M5) mAChR subtypes. Two of the three odd-numbered mAChRs (M1, M5) were present on the hair cell cilia, supporting cells and nerve terminals. And all three odd numbered mAChRs (M1, M3 and M5) were expressed on cuticular plates, myelin sheaths and Schwann cells. Even-numbered mAChRs were seen on the nerve terminals. M2 was also shown on the cuticular plates and supporting cells. Vestibular efferent fibers and terminals were not identified in our studies. Results from WB of the dissociated vestibular epithelia, nerve fibers and vestibular ganglia were consistent with the results from IH and IC. Our findings suggest that there is considerable co-expression of the subtypes on the neural elements of the labyrinth. Further electrophysiological and pharmacological studies should delineate the mechanisms of action of muscarinic acetylcholine receptors on structures in the labyrinth.
Intense sound-induced plasticity in the dorsal cochlear nucleus of rats: Evidence for cholinergic receptor upregulation
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Physiological studies indicate that the AChRs exerting the most influence on spontaneous activity of neurons in the superficial DCN are predominantly muscarinic (mAChRs) (Chen et al., 1994; Chen and Jastreboff, 1995). These receptors are found mainly in the granule cell region of the cochlear nucleus and in the fusiform cell layer of the DCN (Chen et al., 1995; Jin and Godfrey, 2006), which also contains granule cells. The number of mAChRs can be upregulated in these regions of the CN as a consequence of peripheral denervation (Jin and Godfrey, 2006).
Previous studies in a number of species have demonstrated that spontaneous activity in the dorsal cochlear nucleus (DCN) becomes elevated following exposure to intense sound. This condition of hyperactivity has aroused considerable interest because it may represent an important neural correlate of tinnitus. There is some evidence that neurons in the superficial DCN, such as cartwheel, stellate and fusiform cells, may contribute to the level of hyperactivity induced by intense sound, although the relative importance of these different cell types is unknown. In the present study, we sought to determine the effect of intense sound exposure on multiunit spontaneous activity both at the DCN surface and in the fusiform cell layer and to examine the influence of cholinergic input to DCN circuits on the level of activity in the fusiform cell layer. Rats were studied in two groups, one of which had been exposed to a continuous intense sound (10 kHz 127 dB SPL) for 4 h while the other group served as unexposed controls. Between 30 and 52 days post-exposure, recordings of multiunit activity were performed at the DCN surface as well as in the middle of the fusiform cell layer. Changes in fusiform cell layer activity were also studied in response to superficial applications of the cholinergic agonist, carbachol, either alone or following pre-application of the cholinergic antagonist, atropine. The results demonstrated that multiunit spontaneous activity in the rat DCN was generally much higher in both control and exposed animals relative to that which has been observed in other species. This activity was significantly higher at the DCN surface of sound-exposed animals than that of controls. In contrast, hyperactivity could not be demonstrated in the fusiform cell layer of sound-exposed animals. Carbachol administration most commonly caused suppression of fusiform cell layer activity. However, this suppression was considerably stronger in the DCN of sound-exposed animals than in controls. These findings suggest that, hyperactivity at the DCN surface of exposed rats may arise as a consequence of more highly activated neurons in the molecular layer, such as cartwheel and/or stellate cells, and that the lack of hyperactivity in the fusiform cell layer may be the result of inhibition of fusiform cells by these inhibitory interneurons. Although this finding does not rule out fusiform cells as possible sources of hyperactivity in other species, or even in the rat after short post-exposure recovery periods, the enhanced sensitivity of the fusiform cell layer to cholinergic stimulation suggests that in the rat, at least after prolonged post-exposure recovery periods, increased inhibition of activity in this layer by more superficially located neurons may result from an upregulation of receptors for cholinergic input. This upregulation may be greater in rats than in other species due to the relatively heavy cholinergic input that exists in the cochlear nucleus of this species.
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Choline acetyltransferase (ChAT) activity has been mapped in the cochlear nucleus (CN) of control hamsters and hamsters that had been exposed to an intense tone. ChAT activity in most CN regions of hamsters was only a third or less of the activity in rat CN, but in granular regions ChAT activity was similar in both species. Eight days after intense tone exposure, average ChAT activity increased on the tone-exposed side as compared to the opposite side, by 74% in the anteroventral CN (AVCN), by 55% in the granular region dorsolateral to it, and by 74% in the deep layer of the dorsal CN (DCN). In addition, average ChAT activity in the exposed-side AVCN and fusiform soma layer of DCN was higher than in controls, by 152% and 67%, respectively. Two months after exposure, average ChAT activity was still 53% higher in the exposed-side deep layer of DCN as compared to the opposite side. Increased ChAT activity after intense tone exposure may indicate that this exposure leads to plasticity of descending cholinergic innervation to the CN, which might affect spontaneous activity in the DCN that has been associated with tinnitus.
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The determination of the prominence of muscarinic receptor subtypes is limited by the only preferential selectivity of available muscarinic subtype antagonists. However, comparisons of the IC50 values for the muscarinic antagonists with their corresponding Ki values enables the suggestion that, as for the DCN [9], the M2 and M4 subtypes are relatively more prominent than M1 and M3 in the MVN. Although mainly excitatory effects of activation of M2 and M4 receptors in the VNC and DCN would disagree with previous literature suggesting that these muscarinic receptors mediate inhibitory neuronal responses [12], this may be indicative of regional differences in the functions of muscarinic receptor subtypes.
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Muscarinic receptor subtypes in rat dorsal cochlear nucleus

Abstract

Neuron

Neuroscience

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Life Sci.

J. Neurosci. Meth.

Biochem. Pharmacol.

J. Neurosci. Meth.

Life Sci.

Life Sci.

Trends Pharmacol. Sci.

Eur. J. Pharmacol.

J. Biol. Chem.

Brain Res.

Neuroscience

Eur. J. Pharmacol.

A comparison of affinity constants for muscarine-sensitive acetylcholine receptors in guinea-pig atrial pacemaker cells at 29 C and in ileum at 29 C and 37 C

Br. J. Pharmacol.

Distribution and targets of the cartwheel cell axon in the dorsal cochlear nucleus of the guinea pig

Anat. Embryol.

Identification of a family of muscarinic acetylcholine receptor genes

Science

Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-K1 cells

Mol. Pharmacol.

Pharmacology of the putative M4 muscarinic receptor mediating Ca-current inhibition in neuroblastoma × glioma hybrid (NG 108-15) cells

Br. J. Pharmacol.

Effects of cholinergic agonists and antagonists on spontaneous activity of rat dorsal cochlear nucleus neurons: in vitro studies

Soc. Neurosci. Abstr.

Cholinergic effects on spontaneous activity of rat dorsal cochlear nucleus neurons

Abstr. Assoc. Res. Otolaryngol.

Cholinergic modulation of spontaneous activity in rat dorsal cochlear nucleus

Hearing Res.

Glutamate antagonists affect cholinergic activation of bursting neurons in rat dorsal cochlear nucleus

Abstr. Assoc. Res. Otolaryngol.

Muscarinic receptor blockade increases basal acetylcholine release from striatal slices

J. Pharmacol. Exp. Ther.

Antagonist binding profiles of five cloned human muscarinic receptor subtypes

J. Pharmacol. Exp. Ther.

Pharmacology of the putative M₄ muscarinic receptor mediating Ca-current inhibition in neuroblastoma × glioma hybrid (NG 108-15) cells