Glutamic acid decarboxylase levels in the cochlear nucleus of rats with acoustic trauma-induced chronic tinnitus.

Tinnitus is the perception of phantom sounds, a phenomenon believed to be due to abnormal neuronal activity in auditory regions of the CNS such as the brainstem cochlear nucleus (CN). One possible mechanism for the abnormal neuronal activity in the CN, supported by recent animal studies, is a decrease in GABAergic inhibition. One possible explanation for this is a decrease in the enzyme responsible for the synthesis of GABA, glutamic acid decarboxylase (GAD). In this study, we used immunohistochemistry to analyse the levels of GAD in the dorsal and ventral CN of rats that had been exposed to noise trauma and had been confirmed to exhibit psychophysical evidence of tinnitus (at 17.5 weeks post-exposure) using a conditioned behavioural task. At 22 weeks following noise trauma or sham treatment, the levels of GAD in the dorsal and ventral CN were not significantly different. This result suggests that acoustic trauma that can cause chronic tinnitus is not associated with changes in GAD in the CN at 22 weeks post-exposure.

Choline acetyltransferase activity in the hamster central auditory system and long-term effects of intense tone exposure.

Acoustic trauma often leads to loss of hearing of environmental sounds, tinnitus, in which a monotonous sound not actually present is heard, and/or hyperacusis, in which there is an abnormal sensitivity to sound. Research on hamsters has documented physiological effects of exposure to intense tones, including increased spontaneous neural activity in the dorsal cochlear nucleus. Such physiological changes should be accompanied by chemical changes, and those chemical changes associated with chronic effects should be present at long times after the intense sound exposure. Using a microdissection mapping procedure combined with a radiometric microassay, we have measured activities of choline acetyltransferase (ChAT), the enzyme responsible for synthesis of the neurotransmitter acetylcholine, in the cochlear nucleus, superior olive, inferior colliculus, and auditory cortex of hamsters 5 months after exposure to an intense tone compared with control hamsters of the same age. In control hamsters, ChAT activities in auditory regions were never more than one-tenth of the ChAT activity in the facial nerve root, a bundle of myelinated cholinergic axons, in agreement with a modulatory rather than a dominant role of acetylcholine in hearing. Within auditory regions, relatively higher activities were found in granular regions of the cochlear nucleus, dorsal parts of the superior olive, and auditory cortex. In intense-tone-exposed hamsters, ChAT activities were significantly increased in the anteroventral cochlear nucleus granular region and the lateral superior olivary nucleus. This is consistent with some chronic upregulation of the cholinergic olivocochlear system influence on the cochlear nucleus after acoustic trauma.

Calcium-dependent isoforms of protein kinase C mediate glycine-induced synaptic enhancement at the calyx of Held.

Depolarization of presynaptic terminals that arises from activation of presynaptic ionotropic receptors, or somatic depolarization, can enhance neurotransmitter release; however, the molecular mechanisms mediating this plasticity are not known. Here we investigate the mechanism of this enhancement at the calyx of Held synapse, in which presynaptic glycine receptors depolarize presynaptic terminals, elevate resting calcium levels, and potentiate release. Using knock-out mice of the calcium-sensitive PKC isoforms (PKC(Ca)), we find that enhancement of evoked but not spontaneous synaptic transmission by glycine is mediated primarily by PKC(Ca). Measurements of calcium at the calyx of Held indicate that deficits in synaptic modulation in PKC(Ca) knock-out mice occur downstream of presynaptic calcium increases. Glycine enhances synaptic transmission primarily by increasing the effective size of the pool of readily releasable vesicles. Our results reveal that PKC(Ca) can enhance evoked neurotransmitter release in response to calcium increases caused by small presynaptic depolarizations.

Deafferentation-induced redistribution of MMP-2, but not of MMP-9, depends on the emergence of GAP-43 positive axons in the adult rat cochlear nucleus.

The matrix metalloproteinases MMP-9 and MMP-2, major modulators of the extracellular matrix (ECM), were changed in amount and distribution in the rat anteroventral cochlear nucleus (AVCN) following its sensory deafferentation by cochlear ablation. To determine what causal relationships exist between the redistribution of MMP-9 and MMP-2 and deafferentation-induced reinnervation, kainic acid was stereotaxically injected into the ventral nucleus of the trapezoid body (VNTB) prior to cochlear ablation, killing cells that deliver the growth associated protein 43 (GAP-43) into AVCN. Deafferentation-induced changes in the pattern of MMP-9 staining remained unaffected by VNTB lesions. By contrast, changes in the distribution of MMP-2 normally evoked by sensory deafferentation were reversed if GAP-43 positive axons were prevented to grow in AVCN. In conclusion, GAP-43-containing axons emerging in AVCN after cochlear ablation seem to be causal for the maintenance of MMP-2-mediated ECM remodeling.

Bilirubin facilitates depolarizing GABA/glycinergic synaptic transmission in the ventral cochlear nucleus of rats.

Excitotoxicity contributes to bilirubin-induced central nervous system injury; however, the mechanisms involved remain controversial. Previous studies from our lab have demonstrated that in juvenile rats bilirubin facilitates γ-aminobutyric acid (GABA)/glycinergic synaptic transmission through activation of presynaptic protein kinase A (PKA) in isolated neurons of the ventral cochlear nucleus (VCN). However, the descending mechanism and physiological effects of bilirubin-induced potentiation remain unclear. Here, whole-cell recordings show that 3×10(-6) M bilirubin increased the frequency of both spontaneous (sPSCs) and miniature (mPSCs) GABA/glycinergic postsynaptic currents in VCN neurons of postnatal day 12-14 (P12-14) rats. This action was dependent on the concentration and duration of exposure to bilirubin and was only partially suppressed by 10(-5) M bicuculline. The potentiation effect on mPSCs persisted in a Ca2+-free solution, but was fully occluded by pretreatment with 1,2 bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), an intracellular Ca2+ chelator. Following pretreatment of the neurons with BAPTA-AM, forskolin, a PKA activator, had no effect on the frequency or amplitude of mPSCs. This suggests that Ca2+ release from presynaptic stores is part of the descending pathway of PKA activation and is responsible for biliurbin-induced potentiation of cell activity. Using gramicidin-perforated patch recordings, the reversal potential of GABA-evoked currents (EGABA) was also investigated. The GABA response resulted in depolarization of 12 of 20 recorded VCN neurons from P12-14 rats. Therefore, potentiation of depolarizing GABA/glycinergic transmission by bilirubin may underlie bilirubin excitotoxicity, which may play a role in the hearing impairment observed among hyperbilirubinemic neonates.

Afferents to the cochlear nuclei and nucleus laminaris from the ventral nucleus of the lateral lemniscus in the zebra finch (Taeniopygia guttata).

The presence and nature of a descending projection from the ventral nucleus of the lateral lemniscus (LLV) to the cochlear nuclei (NA, NM) and the third-order nucleus laminaris (NL) was investigated in a songbird using tract tracing and GAD immunohistochemistry. Tracer injections into LLV produced anterograde label in the ipsilateral NA, NM and NL, which was found not to be GABAergic. Double retrograde labeling from LLV and NA/NM/NL ruled out the possibility that the LLV projection actually arose from collaterals of superior olivary projections to NA/NM/NL. The LLV projection may be involved in the discrimination of laterality of auditory input.

Olivocochlear neuron central anatomy is normal in alpha 9 knockout mice.

Olivocochlear (OC) neurons were studied in a transgenic mouse with deletion of the alpha 9 nicotinic acetylcholine receptor subunit. In this alpha 9 knockout mouse, the peripheral effects of OC stimulation are lacking and the peripheral terminals of OC neurons under outer hair cells have abnormal morphology. To account for this mouse's apparently normal hearing, it has been proposed to have central compensation via collateral branches to the cochlear nucleus. We tested this idea by staining OC neurons for acetylcholinesterase and examining their morphology in knockout mice, wild-type mice of the same background strain, and CBA/CaJ mice. Knockout mice had normal OC systems in terms of numbers of OC neurons, dendritic patterns, and numbers of branches to the cochlear nucleus. The branch terminations were mainly to edge regions and to a lesser extent the core of the cochlear nucleus, and were similar among the strains in terms of the distribution and staining density. These data demonstrate that there are no obvious changes in the central morphology of the OC neurons in alpha 9 knockout mice and make less attractive the idea that there is central compensation for deletion of the peripheral receptor in these mice.

Evidence for increased NADPH-diaphorase-positive neurons in the central auditory system of the aged rat.

CONCLUSIONS: The age-related increase in the production of nitric oxide (NO) suggests that this increase was related to neuron aging. Additional studies may provide information regarding aging-related changes in the central auditory system. OBJECTIVES: Although NO has been associated with aging, it is unclear whether specific areas of the central auditory system are involved. We therefore assayed aging-related changes in NADPH-diaphorase (NADPH-d), a selective histochemical marker for NO, in the neurons of the central auditory system and other brain regions. MATERIALS AND METHODS: The numbers of NADPH-d-stained neurons and the area and staining density of cell bodies were examined in aged (24 months old) and younger (4 months old) Wistar rats. RESULTS: The number of NADPH-d-positive neurons in the inferior colliculus was significantly increased in aged rats (p<0.05), whereas the area of NADPH-d-positive neurons in all areas did not differ significantly between aged and younger rats (p>0.05). The staining densities of NADPH-d-positive neurons in the inferior colliculus, the auditory cortex, and the visual cortex were significantly greater in aged compared with younger rats (p<0.05).

Regional variations of cytochrome oxidase activity in the central auditory system of Relnrl-Orl (reeler) mutant mice.

Despite preserved cell differentiation, the Reln(rl-Orl) phenotype comprises laminar abnormalities of cell position in auditory cortex and dorsal cochlear nucleus. The metabolic consequences of the cell ectopias were determined by estimating cytochrome oxidase (CO) activity, a marker of neuronal activity. CO activity increased in the granular cell layer of dorsal cochlear nucleus, trapezoid body nucleus, intermediate lateral lemniscus, central and external inferior colliculus, and pyramidal cell layer of primary auditory cortex. On the contrary, CO activity decreased in the superficial molecular layer of dorsal cochlear nucleus as well as in the medioventral periolivary nucleus. These metabolic variations are discussed in terms of their possible relation to morphologic anomalies observed in the mutant.

Neuronal nitric oxide synthase expression in the cochlear nucleus in a salicylate model of tinnitus.

Although a number of studies suggest that the development of tinnitus is associated with hyperactive neuronal discharges in the brainstem cochlear nucleus (CN), there is relatively little evidence to indicate the neurochemical basis of this phenomenon. While some studies suggest that it may be partly due to a decrease in GABAergic inhibition, it is also possible that increased excitability is a contributing factor. In the current study, we investigated whether the salicylate animal model of tinnitus is associated with changes in the number of CN neurons expressing neuronal nitric oxide synthase (nNOS), one of the NOS isoforms that results in the production of the neurotransmitter, nitric oxide. We used a behavioral conditioning paradigm to confirm that animals receiving salicylate injections experienced tinnitus, and used immunohistochemistry with stereology to quantify the number of nNOS-expressing neurons in the dorsal and ventral CN (DCN and VCN, respectively) in salicylate- and vehicle control-treated animals. We also employed Western blotting to quantify the amount of nNOS protein expression in the total CN (i.e., the DCN and VCN together). We found a significant increase (of approximately 70%) in the number of nNOS-expressing principal neurons in the VCN of salicylate-treated animals compared to controls, with no significant differences in the DCN; nor did we find any significant difference in the overall level of nNOS protein in the total CN using Western blotting. These results suggest that changes in the number of neurons in the VCN expressing nNOS may be implicated in the mechanisms of tinnitus.

Effects of intense tone exposure on choline acetyltransferase activity in the hamster cochlear nucleus.

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.

Biotinidase reveals the morphogenetic sequence in cochlea and cochlear nucleus of mice.

Hearing loss affects children with biotinidase deficiency, an inherited metabolic disorder in the recycling of biotin. The deficit appears shortly after birth during development of the auditory system. Using a mouse model, we sought to discover where and when biotinidase is expressed in the normal development of the cochlea and cochlear nucleus. In the process, we reconstructed the normal morphogenetic sequences of the constituent cells. Immunolabeling for biotinidase was localized to neurons and other cells of the adult and immature mouse, including the embryonic precursors of these regions dating from the stage of the otocyst. Its distribution was compared to the particular morphological changes occurring at each developmental stage. Biotinidase was localized in cells and their processes at the critical stages in their proliferation, migration, structural differentiation, and innervation, covering the entire span of their development. The prevalence of immunostaining peaked in the adult animal, including hair cells and ganglion cells of the cochlea and neurons of the cochlear nucleus. The findings suggest that biotinidase plays a role in the normal development of the auditory system. Besides the pattern of localization of biotinidase, this study provides the first systematic account of each developmental stage in a mammalian auditory system.

Effects of cochlear ablation on choline acetyltransferase activity in the rat cochlear nucleus and superior olive.

Using microdissection and quantitative microassay, choline acetyltransferase (ChAT) activity was mapped in the cochlear nucleus (CN) and in the source nuclei of the olivocochlear bundle, the lateral superior olive and ventral nucleus of the trapezoid body. In control rats, gradients of ChAT activity were found within the major subdivisions of the CN and in the lateral superior olive. These gradients correlated with the known tonotopic organizations, with higher activities corresponding to locations representing higher sound frequencies. No gradient was found in the ventral nucleus of the trapezoid body. In rats surviving 7 days or 1 or 2 months after cochlear ablation, ChAT activity was increased 1 month after ablation in the anteroventral CN by 30-50% in most parts of the lesion-side and by 40% in the contralateral ventromedial part. ChAT activity in the lesion-side posteroventral CN was increased by approximately 40-50% at all survival times. Little change was found in the dorsal CN. Decreases of ChAT activity were also found ipsilaterally in the lateral superior olive and bilaterally in the ventral nucleus of the trapezoid body. Our results suggest that cholinergic neurons are involved in plasticity within the CN and superior olive following cochlear lesions.

Contributions of age, cochlear integrity, and auditory environment to avian cochlear nucleus metabolism.

Most commercially raised broiler chickens display progressive cochlear degeneration with age [Hear. Res. 166 (2002) 82]. Recent work examining the effects of age and cochlear degeneration on avian cochlear nucleus (nucleus magnocellularis, NM) metabolism showed that changes in metabolic activity occur with age and cochlear damage [Hear. Res. 175 (2003) 101]. The auditory environment also differed between facilities housing young and adult birds. The relative contributions of age, cochlear degeneration, and auditory environment to these changes in NM metabolism are unknown. Using cytochrome oxidase (CO) histochemistry, NM neuron metabolism is examined in several age groups of birds under varying conditions. When normal cochlear integrity and auditory environment are held constant, CO staining is significantly decreased in adult vs. young birds. When age and auditory environment are held constant, CO staining is significantly decreased in birds with damaged vs. normal cochleae. When age and normal cochlear integrity are held constant, CO staining is significantly decreased in birds living in a quiet vs. noisy environment. All factors examined cause changes in CO staining, which is indicative of NM metabolic activity. Results are discussed in the context of mitochondrial aging, afferent regulation, and auditory deprivation and enrichment.

Co-induction of growth-associated protein GAP-43 and neuronal nitric oxide synthase in the cochlear nucleus following cochleotomy.

In adult animals, cochlear lesioning leads to a reactive synaptogenesis with a reemergence of growth-associated protein, GAP-43, in the auditory brainstem nuclei. In addition, nitric oxide (NO) is also implicated in synaptogenesis. Three isoforms of nitric oxide synthase (NOS) responsible for generating NO have been identified and, in neurons, the predominant isoform is neuronal NOS (nNOS). Studies in visual or olfactory systems have found that the NOS expression often correlates with periods of axonal outgrowth and synapse formation; whether NO plays a similar role in the auditory brainstem needs to be examined. In the present study, a unilateral cochleotomy was performed in adult mice to examine the relationship between the reemergence of GAP-43 and the expression pattern of nNOS. Following surgery, GAP-43 re-emerged in the ipsilateral anterior ventral cochlear nucleus (AVCN) and the immunoreactivity reached a climax around postoperative day (POD) 8; the same expression pattern as that reported in the previous literature is the indicator of synaptogenesis. As for the nNOS immunoreactivity, a dramatic redistribution from a mostly cytoplasmal to a predominantly membranous localization in the ipsilateral AVCN was found especially at POD 4. A similar redistribution pattern in the ipsilateral AVCN for the N-methyl-D-aspartate (NMDA) receptor was also observed at POD 4, corresponding to the fact that the activation of nNOS is coupled to calcium influx via the NMDA-receptor. Furthermore, the expression of cyclic guanosine monophosphate (cGMP) is an indicator for activity of soluble guanylyl cyclase (sGC), the substrate of NO, which reveals the target area of NO. Therefore, cGMP immunoreactivity was also examined and an obvious increase of cytoplasmal cGMP expression was observed around POD 4. Accordingly, it is suggested that nNOS activity correlates closely with the reactive synaptogenesis following a cochleotomy. Further evidence is shown by the results of fluorescent double staining; nNOS-positive cells were surrounded by GAP-43 labeled regions that appeared to be presynaptic boutons, and the vast majority of nNOS-positive cells also expressed cGMP. The former result indicates that, after surgery, there should be new terminal endings projecting onto the nNOS-positive cells in the AVCN. Furthermore, the latter result suggests a possible role of an autocrine mediator for nNOS in the AVCN.

Transient expression of acetylcholinesterase in the posterior ventral cochlear nucleus of rat brain.

In this report we partially characterize a pathway projecting to the posterior ventral cochlear nucleus (PVCN) of the rat brain that transiently expresses a high level of acetylcholinesterase (AChE). The AChE-positive axons form a network that envelops a discrete region of the PVCN that includes the octopus cell region and some cells rostral to it. AChE is first detectable by postnatal day 3 (P3), peaks in expression at about P7-10, and is barely detectable in our preparations by P15. We previously reported that neurons in the octopus cell region express high levels of alpha7 nAChR mRNA and alpha-bungarotoxin binding during the same time period. In light microscopic immunocytochemical studies using antibodies to the vesicular acetylcholine transporter (VAChT), we could not identify immunopositive boutons in the developing regions of the PVCN that express high levels of AChE-positive fibers despite distinct punctate labeling in other brain regions. Systematic electron microscopic examination of AChE histochemical staining throughout the PVCN revealed intense labeling of axons, but synaptic sites were devoid of reaction product. The source of the AChE-positive fibers is not known, but the fibers are not auditory nerve axons and probably not collaterals of the olivocochlear bundle.

Afferent regulation of cytochrome-c and active caspase-9 in the avian cochlear nucleus.

During development, a subpopulation (approximately 30%) of neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), dies following removal of the cochlea. It is clear that neuronal activity coming from the auditory nerve provides trophic support critical for cell survival in the NM. Several aspects of the intracellular signaling cascades that regulate apoptosis have been defined for naturally occurring, or programmed cell death, in neurons. These intracellular cascades involve the extrusion of cytochrome-c from the mitochondria into the cytosol and the subsequent activation of proteolytic caspase cascades, which ultimately act on substrates that lead to the death of the cell. In contrast, the intracellular signaling cascades responsible for deafferentation-induced cell death are not fully understood. In the present series of experiments, the potential extrusion of cytochrome-c from the mitochondria into the cytosol, and the activation of caspases were examined in the NM following deafferentation. Cytochrome-c immunoreactivity increased within 6 h following deafferentation and persisted for at least 3-5 days following surgery. However, cytochrome-c was not detectable within immunoprecipitates obtained from cytosolic fractions of deafferented NM neurons. This suggests that the increased immunoreactivity of cytochrome-c is related to mitochondrial proliferation. As a positive control, cytochrome-c was detected in cytosolic fractions of deafferented NM neurons treated with kainic acid, a substance known to cause cytochrome-c release into the cytosol. In addition, immunoreactivity for downstream active caspase-9 did increase following cochlea ablation. This increase was observed within 3 h following cochlea removal, but was not observed 4 days following surgery, a time point after the dying population of NM neurons have already degenerated. Together, these findings suggest that deafferentation of NM neurons results in caspase activation, but this activation may be cytochrome-c independent.

ERK and SAPK signaling in auditory brainstem neurons after unilateral cochlear ablation.

Unilateral cochlear ablation (UCA) in adults deafferented one cochlear nucleus (CN) and induced several plasticities in central auditory pathways. To assess whether signal transduction could contribute to these changes, we determined if UCA induced activity in the extracellular signal-regulated kinase (ERK) and the stress-activated protein kinase (SAPK) signal transduction pathways. Using Western blots, we measured phosphorylated ERK1 (ERK1-P), ERK2 (ERK2-P), p46 and p54 SAPK (SAPK-P) and c-Jun (c-Jun-P) levels in the major subdivisions of the CN, the principal nuclei of the superior olivary complex (SOC) and the central nucleus of the inferior colliculus (ICc) for up to 145 days postablation. ERK1-P and ERK2-P were typically elevated at 7 and 145 days but depressed at 30 days, 60 days, or both. In addition, ERK1-P and ERK2-P were elevated at 3 days in the anteroventral (AVCN) and posteroventral CN (PVCN). Immunohistochemical labeling indicated that after 5 days, most ERK1/2-P in the CN was in neuronal nuclei. Only minor changes were evident in total ERK1 and ERK2 levels. Several correlations were evident between the postablation plasticities observed previously and altered ERK1-P and ERK2-P levels. Few changes were found in SAPK-P and c-Jun-P levels. Concomitant elevations of SAPK-P and c-Jun-P were not evident, except in the superficial dorsal CN (DCN) at postablation day 3, consistent with a cell-stress response. These findings suggest that signals induced as a consequence of UCA are transduced mainly through the neuronal ERK pathway. This activity probably influenced gene expression and cytoplasmic regulatory mechanisms that contributed to the plasticities induced by UCA.

TrkB levels in the cochlear nucleus after unilateral cochlear ablation: correlations with post-lesion plasticity.

Tyrosine kinase B (TrkB) levels in the adult guinea pig cochlear nucleus (CN) were determined from Western blots for up to 60 days after unilateral cochlear ablation (UCA). Compared to TrkB levels on the intact side, those on the lesioned side were elevated in the anteroventral CN (AVCN) at 7 and 60 days and in the posteroventral CN (PVCN) at 30 days. TrkB levels were depressed in the AVCN and the dorsal CN (DCN) at 30 days. Elevations in the AVCN on the lesioned side at 7 days coincided with a period of synaptogenesis. Other changes were temporally related to up- or downregulations of transmitter release and synaptic receptor activities that were evident after UCA. The findings suggest that changes in signaling through TrkB may contribute to the plasticities in the CN that were evident after UCA.

Protein kinase C mediates potentiation of synaptic transmission by phorbol ester at parallel fibers in the dorsal cochlear nucleus.

Many cells in the outer two layers of the dorsal cochlear nucleus (DCN) express high levels of the phospholipid-activated, calcium dependent kinase, protein kinase C (PKC), an enzyme that can phosphorylate numerous proteins involved in neurotransmission and postsynaptic signaling. We investigated the effects of stimulating PKC with phorbol esters (phorbol 12-13 diacetate; PDAc) on parallel fiber synaptic transmission in brain slices of the guinea pig DCN. Phorbol esters increased the amplitude of the postsynaptic components of the field potential, including the excitatory post-synaptic field potential (fEPSP) and the population spike following electric stimulation of parallel fibers. Phorbol esters simultaneously decreased paired-pulse facilitation, suggesting that transmitter release mechanisms were affected. Potentiation of synaptic transmission and diminished paired-pulse potentiation were also observed in intracellular recordings of DCN neurons. The effects of phorbol esters were antagonized by the specific PKC blockers bisindolylmaleimide and calphostin C. Although modulation of the synaptic potentials appears to be mediated by presynaptic PKC, the differential effects of PDAc on the fEPSP and the population spike also suggest the involvement of postsynaptic PKC and postsynaptic targets. These experiments demonstrate that protein kinase C is capable of profoundly modulating synaptic transmission at parallel fiber synapses in the DCN.