GABAergic inhibition upon auditory response properties of neurons in the dorsal cochlear nucleus of the rat

Summary It is well known that the superficial layers of the dorsal cochlear nucleus (DCN) are rich in GABAergic neurons. We investigated the effects of topical application of GABA receptor agonists and/or antagonists upon the auditory response properties of DCN neurons in rats anesthetized with alpha chloralose-urethane. Auditory stimuli consisted of 20 ms tone bursts presented in a free field. Response properties of DCN neurons were studied before and during iontophoretic application of GABA, bicuculline methiodide (BIC) and muscimol (MUS) alone and GABA with MUS or BIC through triple barrel electrodes glued to the recording microelectrode. Of 68 DCN neurons studied, 27 were sensitive to topical application of the GABA agonists or antagonist. In these neurons, BIC enhanced spontaneous activity as well as auditory responses and decreased the Q-30 quality factor values. MUS reduced auditory responses. BIC often increased the width of the tuning curve but GABA and/or MUS reduced it. Without drug application, GABA sensitive neurons tended to have longer response latencies and larger tuning widths at 30 dB above threshold as well as larger Q-30 values as compared with neurons that were insensitive to GABA. These findings suggest that: 1) GABAergic neurons determine the width of the tuning curve in neurons with GABA receptors by curtailing the excitatory response area, and 2) such neurons receive tonic inhibition from intrinsic GABAergic neurons.     

Subsurface and cytoplasmic cisterns associated with mitochondria in pyramidal neurons of the rat dorsal cochlear nucleus

Pyramidal cells, the principal neurons of the dorsal cochlear nucleus in the rat, have a system of hypolemmal cisterns as prominent as that of cerebellar Purkinje cells. In their perikarya nearly all the subsurface cisterns are closely apposed to mitochondria as in Purkinje cells. This feature emphasizes a similarity between the two types of neuron which both have dendrites residing in the molecular layer. In addition, cochlear pyramidal neurons contain a distinct cytoplasmic cysternal core which includes cisternae with narrow lumina. These occasionally form simple and multiple assemblies with mitochondria.     

Inhibitory synaptogenesis in the rat anteroventral cochlear nucleus

Spherical cells in the anteroventral division of the cochlear nucleus, which relay excitatory inputs from the auditory nerve, also receive both GABAergic and glycinergic inhibitory synapses. Inhibition mediated by GABA and glycine fulfils essential roles in the processing abilities of these and other auditory neurons. However, the developmental program leading to a mature complement of GABAergic and glycinergic synapses and microcircuits is largely unknown. Because of their relatively simple geometry, spherical cells provide an excellent model for unraveling basic developmental patterns of inhibitory synaptogenesis. Using a combination of high resolution immunocytochemical methods, we report that, in the rat, synapses containing GABA or glycine are deployed on spherical cell bodies over a time period extending well beyond hearing onset. Such postnatal developmental recruitment of inhibitory endings is progressive, although there are two distinct leaps in their numbers. The first occurs by the end of the first postnatal week, prior to hearing onset, and the second, during the third postnatal week, after hearing onset. This pattern suggests that adjustments in inhibition could be driven by acoustic experience. While GABAergic and glycinergic endings are maturing and growing in number and size, their neurotransmitter content also appears to be developmentally regulated. Quantitative ultrastructural immunocytochemistry with colloidal gold suggests that GABA and glycine accumulation in synaptic endings follows a staggered pattern, with labeling stabilizing at adult levels by postnatal day 21. This may account for adjustments in synaptic efficacy and strength.     

Multipolar cells in the ventral cochlear nucleus project to the dorsal cochlear nucleus and the inferior colliculus

When retrograde markers are placed in the dorsal cochlear nucleus two classes of labeled cells are found in the ventral cochlear nucleus. These are multipolar cells and granule cells. The structure and distribution of labeled multipolar cells greatly resemble those seen following injection of retrograde markers into the contralateral inferior colliculus. When one retrograde marker is placed in the dorsal cochlear nucleus and another simultaneously placed into the contralateral inferior colliculus, large numbers of multipolar cells containing both markers are found in the ventral cochlear nucleus. These findings show that all or most cells in the ventral cochlear nucleus that project to the inferior colliculus also send collaterals to the ipsilateral dorsal cochlear nucleus.     

Development of oriented dendritic fields in the dorsal cochlear nucleus of the hamster

The shapes of the apical and basal dendritic fields of the fusiform cells in the dorsal cochlear nucleus of the hamster were analyzed quantitatively. Measurements of the cross-sectional areas of the dendritic fields in planes parallel and perpendicular to planes occupied by the cochlear nerve axons revealed that the apical dendrites have a preferential orientation with respect to the cochlear planes. No consistent orientation of the basal dendrites was apparent. The orientation of the apical dendrites is not present in the neonatal hamster; rather, it develops gradually and is not fully established until after postnatal day 25. Since the cochlear fibers grow into the dorsal cochlear nucleus, establish contacts and become functional before day 25, the presence of the cochlear input might be a prerequisite for the development of oriented dendritic trees. In adult hamsters that have had their cochlear input eliminated during infancy, the apical dendritic trees of the fusiform cells are not oriented normally. The results suggest that the presence of the afferent input is necessary for the differential growth that leads to the development of oriented dendritic trees.     

Age-related changes in glycine receptor subunit composition and binding in dorsal cochlear nucleus

Age-related hearing loss, presbycusis, can be thought of, in part, as a slow progressive peripheral deafferentation. Previous studies suggest that certain deficits seen in presbycusis may partially result from functional loss of the inhibitory neurotransmitter glycine in dorsal cochlear nucleus (DCN). The present study assessed age-related behavioral gap detection changes and neurochemical changes of postsynaptic glycine receptor (GlyRs) subunits and their anchoring protein gephyrin in fusiform cells of young (7–11 months) and aged (28–33 months) Fischer brown Norway (FBN) rats. Aged rats showed significantly (20–30 dB) elevated auditory brainstem-evoked response thresholds across all tested frequencies and worse gap detection ability compared to young FBN rats. In situ hybridization and quantitative immunocytochemistry were used to measure GlyR subunit message and protein levels. There were significant age-related increases in the α₁ subunit message with significant age-related decreases in α₁ subunit protein. Gephyrin message and protein showed significant increases in aged DCN fusiform cells. The pharmacologic consequences of these age-related subunit changes were assessed using [³H] strychnine binding. In support of the age-related decrease of α₁ subunit protein levels in DCN, there was a significant age-related decrease in the total number of GlyR binding sites with no significant change in affinity. These age-related changes may reflect an effort to reestablish a homeostatic balance between excitation and inhibition impacting on DCN fusiform cells by downregulation of inhibitory function in the face of an age-related loss of peripheral input. Age-related decrease in presynaptic glycine release results in altered subunit composition and this may correlate with loss of temporal coding of the aged fusiform cell in DCN. The previously reported role for gephyrin in retrograde intracellular receptor subunit trafficking could contribute to the α₁ decrease in the face of increased message.     

Aged-related loss of temporal processing: Altered responses to amplitude modulated tones in rat dorsal cochlear nucleus

Loss of temporal processing is characteristic of age-related loss of speech understanding observed in the elderly. Inhibitory glycinergic circuits provide input onto dorsal cochlear nucleus (DCN) projection neurons which likely serve to modulate excitatory responses to time-varying complex acoustic signals. The present study sought to test the hypothesis that age-related loss of inhibition would compromise the ability of output neurons to encode sinusoidally amplitude modulated (SAM) tones. Extracellular recordings were obtained from young and aged FBN rat DCN putative fusiform cells. Stimuli were SAM tones at three modulation depths (100, 50, and 20%) at 30 dB hearing level with the carrier frequency set to the unit's characteristic frequency. Discharge rate and synchrony were calculated to describe SAM responses. There were significant age-related changes in the shape and peak vector strength [best modulation frequency (BMF)] of temporal modulation transfer functions (tMTFs), with no significant age-related changes in rate modulation transfer functions (rMTFs) at BMF. Young neurons exhibited band-pass tMTFs for most SAM conditions while aged fusiform cells exhibited significantly more low-pass or double-peaked tMTFs. There were significant differences in tMTFs between buildup, pauser-buildup, and wide-chopper temporal response types. Young and aged wide-choppers displayed significantly lower vector strength values than the other two temporal DCN response types. Age-related decreases in the number of pauser-buildup response types and increases in wide-chopper types reported previously, could account, in part, for the observed loss of temporal coding of the aged fusiform cell. Age-related changes in SAM coding were similar to changes observed with receptor blockade of glycinergic inhibition onto fusiform cells and consistent with previously observed age-related loss of endogenous glycine levels and changes in normal adult glycine receptor function. DCN changes in SAM coding could, in part, underpin temporal processing deficits observed in the elderly.     

Pyramidal neurones of the dorsal cochlear nucleus: A golgi and computer reconstruction study in cat

The main projection neurones of the dorsal cochlear nucleus, termed pyramidal, bipolar or fusiform cells, have an apical dendritic arbor approaching the ependymal surface of the nucleus and a basal arbor oppositely directed. In Golgi-Del Rio-Hortega material these neurones were studied, with the light microscope, in nonconventional planes of sectioning oriented across or parallel to the main axis of the elongated nucleus. The pyramidal neurones were seen to be flattened across this axis.The size, shape and orientation of 21 cells from six blocks were studied in detail with computer-aided graphic reconstructions including stereo views. Camera lucida drawings of each cell (usually from several sections) were digitized to obtain x and y coordinates while z coordinates (depths in the tissue) were read from the fine focus knob during microscopy and typed interactively during digitization. The z values were corrected for the effects of refractive index differences in the optical system.Since it was the aim of this study to focus on some fundamental principles of structure and arrangement of pyramidal cells in the dorsal cochlear nucleus rather than on topographic variations, only the middle, regularly built part of the nucleus was examined. Towards the ends of the nucleus the architecture is less regular and will require separate analysis.Measurements of arbor and total cell height and of dendritic length are given. The height of the apical and basal arbor in individual cells showed considerable reciprocity. The total dendritic length was up to 8300 μm (average 6536 μm). The basal arbors always proved to be conspicuously flattened; roughly, the width varied between about 300 and 700 μm (average 489 μm) and the thickness between 65 and 105 μm (average 80 μm). The apical arbors were also often flattened but much less and with a greater variability than the basal arbors (average width 319 μm, thickness 115 μm). The two arbors of individual cells were practically coplanar, the arbor planes showing only moderate angularity (bend) and/or torsion relative to each other (angularity maximum 10°, average 5°; torsion maximum 18°, average 6°). The mutual orientation of cells from the same block was examined. The planes through the basal arbors proved to be very parallel, the differences in orientation angles being between 10 and 0° with rare exceptions. Clearly flattened, apical arbors showed a somewhat greater spread.It may be fundamental for the tonotopic organization of the dorsal cochlear nucleus that a high degree of flatness and parallel orientation are found in the basal arbor which is a target of primary cochlear afferents. Our findings invite detailed charting of these fibers in relation to the arbor planes. Also in electrophysiological studies the planarity and regular orientation of the pyramidal cells should be taken into consideration.     

Cytology, synaptology and immunocytochemistry of commissural neurons and their putative axonal terminals in the dorsal cochlear nucleus of the rat

The first binaural integration within the auditory system responsible for sound localization depends upon commissural neurons that connect the two symmetrical cochlear nuclei. These cells in the deep polymorphic layer of the rat dorsal cochlear nucleus were identified with the electron microscope after injection of the retrograde tracer, Wheat Germ Agglutinin conjugated to Horseradish Peroxydase, into the contralateral cochlear nucleus. Commissural neurons are multipolar or bipolar with an oval to fusiform shape. Few commissural neurons, most inhibitory but also excitatory, connect most of the divisions of the rat cochlear nuclei. The most common type is a glycinergic, sometimes GABAergic, moderately large cell. Its ergastoplasm is organized into peripheral stacks of cisternae, and few axo-somatic synaptic boutons are present. Another type of commissural neuron is a medium-sized, spindle-shaped cell, glycine and GABA-negative, with sparse ergastoplasm and synaptic coverage. A giant, rare type of commissural neuron is glycine-positive and GABA-negative, with short peripheral stacks of ergastoplasmic cisternae. It is covered with synaptic boutons, many of which contain round synaptic vesicles. Another rare type of commissural neuron is a moderately large cell, oval to fusiform in shape, immunonegative for both glycine and GABA, and contacted by many axo-somatic boutons. It contains large dense mitochondria and numerous dense core vesicles of peptidergic type. Some labelled boutons, mostly inhibitory and probably derived from commissural neurons, contact pyramidal, cartwheel, giant and tuberculo-ventral neurons. The prevalent inhibition of electrical activity in a cochlear nucleus observed after stimulation of the contralateral cochlear nucleus may be due to commissural inhibitory terminals which contact excitatory neurons such as pyramidal and giant cells. Other inhibitory commissural terminals which contact inhibitory neurons such as cartwheel and tuberculo-ventral neurons, may explain the stimulation of electrical activity in the DCN after contralateral stimulation.     

大鼠中缝大核向脊髓投射神经元与神经降压素能终末的突触联系

神经降压素(NT)是中枢下行抑制系统的重要神经活性物质。本研究运用免疫组织化学与逆行追踪法相结合的双标技术,在电镜下观察NT能终末与中缝大核(NRM)向脊髓投射神经元的突触联系。在光镜下可见NT阳性纤维和终末散在分布于NRM,但未见NT阳性神经元;将HRP注入腰髓背角后,在NRM内可见比较密集的HRP逆标神经元。在电镜下可见NT阳性终末与HRP逆标神经元的胞体和树突形成以非对称性为主的轴-体突触和轴-树突触。上述结果说明NT可能调控NRM向脊髓背角投射神经元的活动,借此对伤害性信息向中枢的传递发挥抑制效应。     

Tinnitus suppression by electrical stimulation of the rat dorsal cochlear nucleus

Previous studies indicate that the dorsal cochlear nucleus (DCN) may serve as a generator and/or modulator of noise-induced tinnitus. This prompted an interest to investigate the modulatory role of the DCN in tinnitus suppression. In this study, we chronically implanted the DCN of rats with behavioral evidence of intense tone-induced tinnitus. Behavioral evidence of tinnitus was measured using a gap detection acoustic startle reflex paradigm. Our results demonstrated that electrical stimulation of the DCN suppressed behavioral evidence of tinnitus, especially at high frequencies. The data suggest that the DCN may be used as a target to suppress tinnitus through a bottom-up neuromodulation approach. The underlying mechanism of DCN-stimulation-induced tinnitus suppression was discussed by comparing it with other stimulation modalities.     

大鼠中缝背核一氧化氮合酶神经元投射纤维在大脑皮质微血管的分布

目的：研究通过损毁脑干中缝背核(DR)，探讨中缝背核NOS阳性神经元是否投射分布于大脑皮质微血管。方法：将16只SD雄性成年大鼠分为实验组与对照组。对实验组大鼠中缝背核微量注射喹啉酸，饲养1w，灌注固定，然后将大脑及脑干作冠状冰冻切片，NADPH—d组化染色。结果：实验组大鼠的中缝背核被有效损毁，其NOS阳性神经元的数量减少了59．1％(P＜0．001)。额、顶叶皮质NOS阳性纤维终末减少了32．1％(P＜0．05)，其中附着于皮质微血管的NOS阳性纤维终未了减少了37．8％(P＜0．01)。而枕额叶皮质NOS阳性纤维终末也减少了32．8％(P＜0．05)，其中附着于皮质微血管的阳性纤维终末减少了39．4％(P＜0．01)。结论：位于中缝背核的NOS阳性神经元投射分布于大脑皮质微血管，可能参与大脑皮质血流量的调节。     

Mechanisms of modulation of pregnanolone on glycinergic response in cultured spinal dorsal horn neurons of rat

The glycine receptors and neurosteroids in spinal cord are both implicated in nociceptive signal processing. However, the modulatory effects of neurosteroid pregnanolone (5beta-pregnan-3alpha-ol-20-one) on native glycine receptors remain unclear. In the present study, we examined the effects of pregnanolone and its three isomers on glycine receptors by using whole-cell patch-clamp technique. Our results showed that pregnanolone reversibly inhibited the amplitude of glycine-induced current mediated by native glycine receptors and recombinant alpha1-, alpha2-, alpha3- and alpha1beta-glycine receptors. In cultured spinal dorsal horn neurons of rats, pregnanolone inhibited the glycine-induced current in dose-dependent manner, with an antagonist concentration inducing half-maximal response of 1.0+/-0.3 microM. The inhibitory effect of pregnanolone on glycine-induced current was voltage-independent and pregnanolone shifted the concentration-response curve for glycine-induced current rightward in a parallel manner without altering the maximal value and Hill coefficient. The isomer of pregnanolone, allopregnanolone (5alpha-pregnan-3alpha-ol-20-one) slightly enhanced glycine-induced current, whereas iso-pregnanolone (5beta-pregnan-3beta-ol-20-one) and iso-allopregnanolone (5alpha-pregnan-3beta-ol-20-one) did not affect the glycine-induced current significantly in cultured spinal dorsal horn neurons. Thus, our results suggest that the inhibitory effect of pregnanolone on glycine-induced current is of a competitive type and depends on the stereo structure of pregnanolone. Furthermore, pregnanolone decreased the amplitude and frequency of the glycinergic miniature inhibitory postsynaptic currents. Through modulating the glycinergic inhibitory neurotransmission, pregnanolone may affect the nociceptive sensory processing under physiological and pathological conditions.     

GABA-immunoreactive neurons in the dorsal lateral geniculate nucleus of the rat: characterisation by combined Golgi-impregnation and immunocytochemistry

Summary Identified neurons in the dorsal lateral geniculate nucleus (dLGN) of the rat were studied using a Golgi/EM procedure in combination with post-embedding GABA immunocytochemistry. The results of this investigation identify clearly the GABA-immunoreactive nature of the two morphologically] distinct neuronal populations found within the rat dLGN — thalamocortical relay cells are GABA-immunonegative whilst the putative interneurons are GABA-immunopositive. This immunocytochemical evidence strongly suggests that interneurons synthesize and use GABA to mediate their local inhibitory synaptic mechanisms within the dLGN of the rat.     

Fine structure and neurotransmitter cytochemistry of neurons in the rat ventral cochlear nucleus projecting to the ipsilateral dorsal cochlear nucleus

The neural tracer wheat germ agglutinin conjugated to horse radish peroxidase was injected into the rat dorsal cochlear nucleus and acoustic stria. Some labelled neurons in the ipsilateral ventral cochlear nucleus were found as a result. These neurons were studied at the ultrastructural level, and their axo-somatic synaptic profile and glycine immunoreactivity were determined. Most neurons were glycine negative and classified as type I multipolar neurons. The latter showed a different synaptic profile from that of neurons projecting to the contralateral inferior colliculus or cochlear nucleus. This suggests the presence of differing populations of multipolar cells based on their synaptic profile. Few labelled multipolar neurons of type II were found, which appeared glycine negative and, rarely, glycine positive. The latter show an ultrastructure and axo-somatic profile similar to that of glycinergic commissural neurons in the dorsal and ventral cochlear nucleus. In particular, about one-third of boutons contained round synaptic vesicles, which are believed to contain an excitatory neurotransmitter. The ultrastructural analysis of the synaptic boutons in the cochlear nucleus confirms the presence of numerous cases of colocalization of glycine and GABA where flat and pleomorphic synaptic vesicles are mixed. The present study is in accordance with previous tract-tracing light microscopic studies which have indicated that large glycinergic neurons in the ventral cochlear nucleus act as broad-band inhibitory neurons in microcircuits of the dorsal cochlear nucleus and contralateral cochlear nucleus.     

Radioautographic study of serotoninergic axon terminals in the rat trigeminal motor nucleus

The responses of center red ON and center red OFF ganglion cells of the isolated goldfish retina to a flickering stimulus light diminish by GABA. Without stimulation GABA blocks the maintained activity of the OFF cells. With a green flickering spot in the receptive field center, the green ON-response of the OFF cells becomes an OFF-response during GABA administration. It is concluded that, besides the common antagonistic green input, the receptive field center of these cells also has a synergistic green input, which is mediated by a pathway other than the center opponent red and green inputs.     

Plasticity at glycinergic synapses in dorsal cochlear nucleus of rats with behavioral evidence of tinnitus

Fifteen percent to 35% of the United States population experiences tinnitus, a subjective “ringing in the ears”. Up to 10% of those afflicted report severe and disabling symptoms. Tinnitus was induced in rats using unilateral, 1 h, 17 kHz-centered octave-band noise (116 dB SPL) and assessed using a gap-startle method. The dorsal cochlear nucleus (DCN) is thought to undergo plastic changes suggestive of altered inhibitory function during tinnitus development. Exposed rats showed near pre-exposure auditory brainstem response (ABR) thresholds for clicks and all tested frequencies 16 weeks post-exposure. Sound-exposed rats showed significantly worse gap detection at 24 and 32 kHz 16 weeks following sound exposure, suggesting the development of chronic, high frequency tinnitus. Message and protein levels of α_1–3, and β glycine receptor subunits (GlyRs), and the anchoring protein, gephyrin, were measured in DCN fusiform cells 4 months following sound exposure. Rats with evidence of tinnitus showed significant GlyR α₁ protein decreases in the middle and high frequency regions of the DCN while α₁ message levels were paradoxically increased. Gephyrin levels showed significant tinnitus-related increases in sound-exposed rats suggesting intracellular receptor trafficking changes following sound exposure. Consistent with decreased α₁ subunit protein levels, strychnine binding studies showed significant tinnitus-related decreases in the number of GlyR binding sites, supporting tinnitus-related changes in the number and/or composition of GlyRs. Collectively, these findings suggest the development of tinnitus is likely associated with functional GlyR changes in DCN fusiform cells consistent with previously described behavioral and neurophysiologic changes. Tinnitus related GlyR changes could provide a unique receptor target for tinnitus pharmacotherapy or blockade of tinnitus initiation.     

Ultrastructural study of cholecystokinin-like immunoreactive nerve terminals in the rat nucleus accumbens

The cholecystokinin (CCK)-like immunoreactive nerve terminals were studied in the caudal and medial parts of the rat nucleus accumbens (NA), using the indirect immunoperoxidase technique, at the electron microscopic level. In the labelled axon terminals the immunoprecipitate is localized inside large dense-cored vesicles which are occasionally present, and surrounds small and medium-sized, round, clear synaptic vesicles. The immunoreactive nerve terminals participate in synapses of both asymmetrical and symmetrical types containing mostly small synaptic vesicles. The asymmetrical synapses are much more numerous and mainly axo-spinous. The symmetrical synapses are less frequent and are axo-dendritic or axo-somatic.     

Increases in spontaneous activity in the dorsal cochlear nucleus of the rat following exposure to high-intensity sound

The effects of intense sound exposure on neural activity in the dorsal cochlear nucleus (DCN) were studied in the rat. Seventeen anesthetized adult rats were exposed to a 10-kHz tone at 125–130 dB SPL for 4 h. Fourteen unexposed rats served as controls. Spontaneous activity (SA) and neural thresholds at the characteristic frequency were measured in three rows of 8–12 sites along the mediolateral, tonotopic, axis of the DCN surface 27–61 days after exposure. The results showed that intense tone exposure induced chronic increases in SA. This hyperactivity was found to be distributed broadly across the DCN with an emphasis around the 10-kHz locus and was associated with shifted response thresholds. These findings demonstrate the usefulness of the rat for studies of physiological phenomena related to noise-induced tinnitus and hearing loss.