| Part ICorticofugal Modulation on Acoustically Induced Fos Expression in the Rat Auditory PathwayThe auditory thalamus which includes the medial geniculate body (MGB) and the lateral part of the posterior nucleus group relays information from the inferior colliculus (IC) to the cortex. The MGB is the principal auditory thalamic nucleus, which plays an essential role in the processing of sound. In addition to the ascending projection from the thalamus to the cortex, the MGB receives a much larger reciprocal projection from the cortex. The corticofugal projection, which projects not only to the MGB but also to the IC and brainstem, has been suggested to provide a gating or gain control mechanism in the transmission of information from the periphery to the cortex. In the auditory system, corticofugal modulation on the MGB and IC is tonotopically organized. Anatomically, the majority of corticofugal projections terminate at the thalamus, while a small portion of such fibers terminate at the IC. Most investigations on corticofugal modulatory effects have been carried out with in-vivo electrophysiological recording of MGB and IC neurons while at the same time the auditory cortex was either activated with electrical/chemical stimuli or inactivated by cooling.Neuronal expression of Fos protein following novel physiological input constitutes a useful marker for polysynaptic activation, allowing neuronal subpopulations in specific neuronal circuits to be identified. Fos-immunoreactivity has also been used to identify functionally activated neurons in the ascending auditory system. Nevertheless, there is no available literature on the contribution of the corticofugal projection to neurons in the ascending auditory circuitry.In the present study, we investigated the corticofugal modulation of acoustic stimulus in neurons along the ascending auditory pathway, using immunohistochemical detection of Fos as an indicator of postsynaptic activation. We microinjected bicuculline methobromide (BIM), an antagonist of GABAA receptor, to the auditory cortex to suppress the activity ofinhibitory neurons resulted in hyperactivity of the cortex. Meanwhile, we also investigate the relationships between the c-fos expression in the auditory thalamus and 1) the corticofugal activation, 2) receptors of neurotransmitters, 3) involvement of the thalamic reticular nucleus (TRN), and 4) the neuronal firing patterns with in-vivo multichannel extracellular recording.Specificity of the Fos antibodyTo assess if the antibody PC38 (rabbit polyclonal anti-serum against a synthetic N-terminal fragment at residues 4-17 of human Fos) is specifically reactive to Fos protein expressed in MGB of rats in which the right auditory cortices had been stimulated with BIM, freshly dissected MGB was tested by western blotting experiment. The result showed that c-Fos immunopositivities was selectively neutralized by pre-incubation of extracts with the immunizing Fos peptide.Activation of auditory cortex by bicucullineTo activate the auditory cortex, we injected 0.3 ul BIM into the right auditory cortex. The result showed that this dose of BIM was effective in activating the whole auditory cortex with minimum spread beyond the auditory cortex. Both layers VI and V, which project to subcortical structures, were activated. However, the contralateral auditory cortex was not activated with this amount of BIM injection.Effect of different anestheticTo elucidate the effect of different anesthetic on the expression of Fos, two anesthetic were used at the same experimental condition. The result showed that no difference was found between the animals which received ketamine/xylazine and pentobarbital sodium after they were injected with BIM in the cortex and exposed to the repeated noise-burst stimulus.Fos expression in the MGB triggered by the cortical activationFor each of different acoustic stimulus groups, two controls were performed in accordance with the experimental condition. In the first control of which no cortical injection was made, no Fos expression in the MGB was observed with acoustic stimulation. In the secondcontrol of which saline was injected into the auditory cortex, no Fos-labeled neurons were found in the ventral and dorsal divisions of the MGB on both sides, though a few were detected in the MGm. Our findings indicate that the MGv and MGd exhibited no Fos expression if the anesthetized animals were exposed to sound stimulation but without cortical activation. When the auditory cortex was activated, Fos-positive neurons were found in the ipsilateral side of ventral (MGv), medial (MGm), and dorsal divisions (MGd) of MGB.Fos expression elicited by glutamate but not acetylcholineDirect injection of glutamate in the auditory thalamus elicited Fos expression in the thalamocortical projection neurons, while injection of acetylcholine evoked no Fos expression in the MGv. The induction of Fos expression in the MGB by direct glutamate injection depended on the level of anesthesia: the subjects which were not injected further anesthesia after the procedure showed Fos expression as in Figure 4A, while those which were continuously injected with further anesthesia during their survival period showed no Fos expression in the MGB.In all subjects after BIM injection in the auditory cortex, the MGB showed a heavy labeling of Fos-positive neurons. Antagonists of all three types of glutamate receptors: AP-V, CNQX and LY36385 (Tocris, UK) showed a strong suppression effect on the Fos expression elicited by the corticofugal activation (BIM injection in the auditory cortex). The injection of AP-V, the NMDA receptor antagonist, shown in Figure 5C, located in the caudal part. The suppression of the Fos expression in the middle and rostral MGB was relatively weaker than that by the injection of AMPA and mGlu receptor antagonists. All three antagonist of glutamate receptors had a determine effect in eliminating the Fos expression in the MGv.Activation of the auditory cortex triggered thalamocortical synchronizationWe have monitored the neural activities of the thalamus and cortex through multi-channel extracellular recording (before, during, and after the BIM injection in the auditory cortex). Spontaneous neuronal activities were observed from all recordings in the MGB and the auditory cortex and were not well synchronized before BIM injection in the cortex. During the process of BIM injection in the cortex, it took about 67.8±31.2 seconds (Mean±SD, n=9)for the auditory cortex to start an oscillatory activity, while activities of the thalamus were still not synchronized with the cortex. It took 6.8±3.9 min (n=13) for the cortex and thalamus to develop the synchrony. The right panel of Figure 6A shows the neuronal activities of the thalamus and cortex at 12 min after the BIM injection. In this case, the cortex and thalamus developed a highly synchronized activity with a rhythm of 2.8 Hz, and the time lag between the cortex and thalamus in this case was less than 50 ms. From the depicted traces as shown at the lower row, it was found that the bursts in the cortex came before the thalamus. This phenomenon was confirmed in all subjects with a rhythm of 0.3-3 Hz.Three (Thl-3) of four electrodes in the thalamus were located in the MGB as all of them responded to acoustic stimulus as well as the electrode in the cortex (Figure 6B).Fos expression in the MGBFor each of different acoustic stimulus groups, two controls were performed in accordance with the experimental condition. In the first control of which no cortical injection was made, no Fos expression in the MGB was observed with acoustic stimulation. In the second control of which saline was injected into the auditory cortex, no Fos-labeled neurons were found in the ventral and dorsal divisions of the MGB on both sides, though a few were detected in the MGm. Our findings indicate that the MGv and MGd exhibited no Fos expression if the anesthetized animals were exposed to sound stimulation but without cortical activation. When the auditory cortex was activated, Fos-positive neurons were found in the ipsilateral side of ventral (MGv), medial (MGm), and dorsal divisions (MGd) of MGB.Fos expression in MGv elicited by pure tonesPure tones of different frequencies elicited Fos-positive neurons in all divisions of the MGB.After 1 hour 200 Hz stimulation, Fos-positive neurons were found in the middle portion of the MGv spanning rostrocaudally for 1.2 mm. Labeling was less dense in the rostral and caudal ends of the MGB. After 1 hour 1 kHz stimulation, the distribution of Fos-positive neurons was similar to that of 200 Hz stimulation. Labeling in the caudal part of the MGv tended to be more ventral as compared with that elicited by 200 Hz. After 3 kHz stimulation, Fos-positive neurons assembled more rostrally as compared with those elicited by 1 kHzstimulation. No Fos labeling was found caudal to Bregma -5.96 mm with 3 kHz stimulation though Fos labeling was evidenced after both 200 Hz and 1 Hz stimulations. After 10, 12.5 and 15 kHz stimulations, Fos-positive neurons assembled more rostrally forming a band which extended along the medioventral to dorsolateral dimension.From all six groups, Fos-positive neurons could be detected at the medial and dorsal divisions of the MGB. The distribution pattern of Fos labeling in the MGm and MGd was independent of the frequency of stimulation.Fos-positive neurons in the inferior colliculusNo obvious spatial difference of Fos-positive neurons was found on both sides of the IC when the animals were exposed to 1 hour pure tone of different frequencies after the right auditory cortex was activated with BIM.The number of labeled neurons in the DCIC ipsilateral to the injected cortex was almost doubled that in the DCIC contralateral to the injected cortex (P<0.05, t-test). Fos-labeled neurons in the ECIC ipsilateral to the injected cortex was also significantly more than those in the ECIC contralateral to the injected cortex (P<0.05, t-test). However, no lateral difference in the Fos expression was found in the CIC in the same subject (t-test). No statistical differences were found in any subnuclei of IC among different stimulus frequencies (ANOVA).Fos-positive neurons in the superior olivary complex and cochlear nucleusA small difference in Fos expression to difference stimulus frequencies was found in the lateral superior olivary nucleus (LSO). High frequency evoked Fos-positive neurons were in the dorsolateral LSO, while lower frequency evoked Fos-positive neurons were in the ventral LSO. No statistical difference was found between the LSOs ipsilateral and contralateral to the injected cortex (t-test) and among different frequency groups (ANOVA). No statistical difference of Fos expression in the LSO was found between animals with and without BIM injection (t-test).Fos-positive neurons were found in deeper layer of the cochlear nucleus of all rats only by pure tone stimulation. When the auditory cortex was activated by BIM, acoustic stimulus evoked Fos-positive neurons were found in the superficial layer of dorsal cochlear nucleus (DCN). When the auditory cortex was activated by BIM, high frequency pure tone stimulus (10 kHz or higher) evoked Fos-positive neurons in the granule cell lamina, which was located between the dorsal and ventral CNConclusion1. No Fos-labeled neurons were found in the MGv with acoustic stimuli alone suggests that the transmission of ascending thalamocortical information is critically governed by corticofugal modulation.2. Injection of glutamate neurotransmitter directly to the thalamus caused Fos expression when the animal was allowed to wake up, indicting that glutamate was involved in the corticofugal activation as well as Fos expression in MGv.3. Fos expression in the MGB has a link to the NMDA, AMPA, and mGlu receptors of glutamate, but not to receptors of Ach.4. We would conclude that cortically derived highly synchronized oscillation (1-3 Hz) induced Fos expression in the corticothalamic network, including MGv, in which the primary ascending excitation could not induce Fos expression.5. The location of Fos-labeled neurons in the MGv after acoustic stimulation at different frequencies was in agreement with the known tonotopic organization.6. The dorsal (DCIC) and external cortices (ECIC) of the IC ipsilateral to the BIM-injected cortex showed a significantly higher number of Fos-labeled neurons than the contralateral IC. However, no difference in the number of Fos-labeled neurons was found between the central nucleus of the IC on either side, indicating that direct corticofugal modulation only occurs in the ECIC and DCIC.Part IITime Course of the Denervation Hypersensitivity of the Corticothalamic and Corticocollicular Circuits after Bilateral Cochlear AblationThe major auditory thalamus, the medial geniculate body (MGB), can be divided into the ventral (MGv), dorsal (MGd) and the medial (MGm) divisions. The MGd projects to areas surrounding the primary auditory cortex (AI) while the MGm projects to the entire auditory cortex. Neurons in the MGd and MGm show relatively long response latencies, broad/multi-peaked tuning curves, nontonotopic organization, tonic response pattern, and multi-modality responses. In contrast to the MGd and MGm, neurons in the MGv project to AI and show short latency response, sharp tuning curves and tonotopic organization. Based on its morphology, MGv is further parceled into the par ovoid (OV) and par latelalis (LV) nuclei. Romanski and Ledoux (1993) showed that OV projects purely to AI, while LV projects to AI and surrounding auditory cortex. Physiologically, neurons in these two subnuclei exhibit similar response properties, and have been thought to be complementary to each other.The MGB and inferior colliculus (IC) receive massive feedback projections from the cortex. Cortical feedback to the thalamus has been suggested to exert a gain-control effect on the transmission of sensory information. The lemniscal (i.e. MGv) and non-lemniscal MGB differ in terms of (1) firing patterns (single spikes versus spike bursts, respectively), (2) corticofugal modulations on the auditory ON responses (facilitatory versus inhibitory, respectively), and (3) oscillatory properties (less-oscillatory versus oscillatory, respectively)After losing part of their inputs, central neurons show denervation hypersensitivity as they become more dependent on the remaining inputs. Unilateral cochlear ablation has been shown to result in the weakening of inhibitory input and strengthening of excitatory response in the contralateral IC, indicating a hypersensitivity to the remaining cochlea. After bilateral cochlear ablation, neurons in the MGB, IC, superior olivary complex, and cochlear nucleus showed denervation hypersensitivity to electrical stimulation of the respective nuclei.Fos-immunoreactivity has also been successfully used to identify neurons in the auditory system. In the present study, we used Fos expression as an indicator of polysynapticactivation to assess the hypersensitivity of MGB and IC to cortical input at different time after bilateral cochlear ablation.Fos expression in the MGv of the controlsIn the sham control experiment, the subjects were sacrificed at different survival time after bilateral cochlear ablation but with no injection in the cortex. No Fos-positive neurons were found in the ventral and dorsal divisions of the MGB on both sides, though a few were detected in the MGm. This sham control results were different from those of the experimental animals (i.e. injected of BIM to the auditory cortex at different time after bilateral cochlear ablation) in which all the ventral, dorsal (MGd) and medial divisions (MGm) of the MGB showed strong labeling of Fos-positive neurons. The results of the vehicle controls were the similar with that of sham controls.We designed another two control animals (1 in the C-tb group and the other in the C-ab group). Both of them were injected with BIM in their right auditory cortices immediately after damage of the tympanic membrane or cochlea. Fos-positive neurons were found in the MGv (C-tb, 1631±64.00, n=2; C-ab, 1250±68.50, n=2), MGm (C-tb, 381±35.00, n=2; C-ab, 235.5±34.50, n=2) and MGd (C-tb, 168.5±4.500, n=2; C-ab, 341.0±14.00, n=2).Fos expression in the MGBIn the auditory thalamus, Fos-positive neurons were detected only at the MGB ipsilateral to the BIM-injected cortex. However, the Fos labeling within the ipsilateral MGB of different experimental groups was significantly different. More Fos-positive neurons were labeled in animals with shorter survival periods after bilateral cochlear ablation (ab4hr group: 4,866±407.6, ab24hr group: 5,021±320.0, ab7d group: 3,899±397.9, abl4d group: l,007±36.77, and ab30d group: 1,076±120.0). The number of Fos-positive neurons in the ab4hr and ab24hr groups were significantly higher than those of control groups C-tb (2180±95.00) and C-ab (1826±117.0) (P<0.01).Fos expression in the MGvIn the ab4hr group, Fos-positive neurons were detected mainly in the OV (4,186±360.7, n=5;)and marginally in LV (68.2±20.71, n=5;) of the MGv. The number of Fos-positive neurons in the OV was significantly higher than those of control groups C-tb (1527±45.50) and C-ab (1061±98.08) (P<0.001), while that in the LV was similar to those of these control groups (P>0.05). In the ab24hr group, the number of Fos-positive neurons in the OV (764.4±155.8, n=7) decreased drastically to < 20% of the ab4hr group (P<0.001), while those in the LV (3,531±147.8, n=7) increased greatly by 50 folds (P<0.001, compared with both control groups C-tb and C-ab, and the experimental group ab4hr). In the ab7d group, very few Fos-positive neurons (109.6±25.69, n=5) could be detected in the OV and the number of these neurons was significantly smaller than those of control group C-tb (P<0.01), and the experimental group ab4hr (P<0.001). On the other hand, the number of Fos-positive neurons in the LV (2,991±387.9, n=5) was similar to that of the ab24hr group (P>0.05) but was significantly higher than those of both control groups C-tb and C-ab, and the experimental group ab4hr (P<0.001). Similar to the ab7d group, very few neurons in the OV were labeled in both the ab14d (98.75±6.125, n=4) and ab30d groups (119.3±17.90, n=4). These were significantly fewer than the C-tb group and ab4hr group. The Fos-positive neurons in the LV were also significantly decreased in the ab14d group (574.3±30.68, n=4; P<0.001, compared with both the ab24hr and ab7d groups) and the ab30d group (574.3±78.61, n=4; P<0.001, compared with both the ab24hr and ab7d groups).Fos expression in MGmIn the MGm ipsilateral to the cortex where BIM was injected, similar number of Fos-positive neurons were found among the three experimental groups: ab4hr, ab24hr and ab7d (499.2±84.91, 497.1±75.88, and 536.2±62.70 respectively). The number of Fos-positive neurons decreased significantly in the ab14d (99.75±20.18) and ab30d groups (178.3±26.24) (both P<0.05). In the contralateral MGm, the number of Fos-positive neurons of all groups was low, ranging between 87.5±52.5 and 111.2±12.5.In the control C-th/C-ab groups, only a few Fos-positive neurons were expressed in the MGm.In the vehicle control groups, no difference was found between the MGB on the two sides.Fos expression in MGdWhen compared with other thalamic subnuclei, neurons in the MGd were least labeled.Fos-positive neurons were found between the middle part (-5.50 mm from bregma) and rostral end of the MGB. In the ab4hr group, the number of Fos-positive neurons (112.0±19.75, n=5) was 2.39% of all Fos-positive neurons within the auditory thalamus. As compared to the ab4hr group, Fos-positive neurons increased significantly (P< 0.05) in the ab24hr and ad7d groups (227.9±24.24, n=7 and 262.6±27.86, n=5,). Comparable number of Fos neurons was found in groups abl4d (233.8±10.91, n=4) and ab30d (204.5±35.53, n=4). Since the total number of Fos-positive neurons in MGB decreased drastically (from 4866 ± 407.6, n=5, in ab4hr to 1007±36.7, n=4, in ab14d and 1076±120.0, n=4, in ab30d; both p<0.001), their proportion increased significantly (from 2.39% in ab4hr group to 23.35% and 18.77% respectively).Fos-positive neurons in the inferior colliculusIn contrast to the MGB, Fos-positive neurons were found in all IC subnuclei in all control groups (data not shown). The number of Fos-positive neurons in the IC contralateral to the BIM-injected cortex was similar among all experimental groups. A significantly higher number of Fos-positive neurons were found in the ipsilateral IC suggesting a stronger corticocollicular effect. The Fos-positive neurons in the ipsilateral IC, however, show a decrease trend from 410.8±47.10 in the ab4hr group to 258.3±31.72 (n=4) in the abl4d group and 305.3±35.11 (n=4) in the ab30d group.In the dorsal cortex (DCIC) and external cortex (ECIC) of the IC in all groups, a significant increase in Fos-positive neurons was found in the hemisphere ipsilateral to the BIM-injected cortex when compared with the contralateral hemisphere.In the vehicle control groups, no difference was found between the bilateral ICs. The number of Fos-positive neurons in either side of IC in the vehicle controls was comparable to that in the IC contralateral to the BIM-injected cortex of the experimental groups.Conclusion1. The time course of Fos expression in individual central auditory relays may reflect the order of dependence of different nuclei on the auditory ascending input. Thus, OV should be the first-order nucleus for relaying the ascending auditory information, LV the second-order nucleus, and MGd the third-order nucleus.2. While earlier electrophysiological studies reported that neurons in the OV and LV had similar properties, recent electrophysiology results in the guinea pigs, morphological difference and the present results suggest that OV and LV are functionally segregated.3. The time window of the rise and decay of Fos expression in different MGB nuclei following cortical activation at different time after bilateral cochlear ablation possibly indicates the denervation hypersensitivity and its dependence on corticofugal input. The present study shows for the first time that denervation hypersensitivity could be associated with Fos expression.4. The ipsilateral CIC showed a significant increase in Fos-positive neurons over the contralateral CIC at 4 hr, 1 d, and 7 d after cochlear ablation. This suggests the strong dependence of the CIC on primary ascending auditory input.
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