Age-related upregulation of dense core vesicles in the central inferior colliculus.

Presbycusis is one of the most prevalent disabilities in aged populations of industrialized countries. As we age less excitation reaches the central auditory system from the periphery. To compensate, the central auditory system [e.g., the inferior colliculus (IC)], downregulates GABAergic inhibition to maintain homeostatic balance. However, the continued downregulation of GABA in the IC causes a disruption in temporal precision related to presbycusis. Many studies of age-related changes to neurotransmission in the IC have therefore focused on GABAergic systems. However, we have discovered that dense core vesicles (DCVs) are significantly upregulated with age in the IC. DCVs can carry neuropeptides, co-transmitters, neurotrophic factors, and proteins destined for the presynaptic zone to participate in synaptogenesis. We used immuno transmission electron microscopy across four age groups (3-month; 19-month; 24-month; and 28-month) of Fisher Brown Norway rats to examine the ultrastructure of DCVs in the IC. Tissue was stained post-embedding for GABA immunoreactivity. DCVs were characterized by diameter and by the neurochemical profile (GABAergic/non-GABAergic) of their location (bouton, axon, soma, and dendrite). Our data was collected across the dorsolateral to ventromedial axis of the central IC. After quantification, we had three primary findings. First, the age-related increase of DCVs occurred most robustly in non-GABAergic dendrites in the middle and low frequency regions of the central IC during middle age. Second, the likelihood of a bouton having more than one DCV increased with age. Lastly, although there was an age-related loss of terminals throughout the IC, the proportion of terminals that contained at least one DCV did not decline. We interpret this finding to mean that terminals carrying proteins packaged in DCVs are spared with age. Several recent studies have demonstrated a role for neuropeptides in the IC in defining cell types and regulating inhibitory and excitatory neurotransmission. Given the age-related increase of DCVs in the IC, it will be critical that future studies determine whether (1) specific neuropeptides are altered with age in the IC and (2) if these neuropeptides contribute to the loss of inhibition and/or increase of excitability that occurs during presbycusis and tinnitus.

Age-related upregulation of perineuronal nets on inferior collicular cells that project to the cochlear nucleus.

Introduction: Disruptions to the balance of excitation and inhibition in the inferior colliculus (IC) occur during aging and underlie various aspects of hearing loss. Specifically, the age-related alteration to GABAergic neurotransmission in the IC likely contributes to the poorer temporal precision characteristic of presbycusis. Perineuronal nets (PNs), a specialized form of the extracellular matrix, maintain excitatory/inhibitory synaptic environments and reduce structural plasticity. We sought to determine whether PNs increasingly surround cell populations in the aged IC that comprise excitatory descending projections to the cochlear nucleus. Method: We combined Wisteria floribunda agglutinin (WFA) staining for PNs with retrograde tract-tracing in three age groups of Fischer Brown Norway (FBN) rats. Results: The data demonstrate that the percentage of IC-CN cells with a PN doubles from ~10% at young age to ~20% at old age. This was true in both lemniscal and non-lemniscal IC. Discussion: Furthermore, the increase of PNs occurred on IC cells that make both ipsilateral and contralateral descending projections to the CN. These results indicate that reduced structural plasticity in the elderly IC-CN pathway, affecting excitatory/inhibitory balance and, potentially, may lead to reduced temporal precision associated with presbycusis.

Age-related Changes of GAD1 mRNA Expression in the Central Inferior Colliculus.

Encoding sounds with a high degree of temporal precision is an essential task for the inferior colliculus (IC) to perform and maintain the accurate processing of sounds and speech. However, the age-related reduction of GABAergic neurotransmission in the IC interrupts temporal precision and likely contributes to presbycusis. As presbycusis often manifests at high or low frequencies specifically, we sought to determine if the expression of mRNA for glutamic decarboxylase 1 (GAD1) is downregulated non-uniformly across the tonotopic axis or cell size range in the aging IC. Using single molecule in situ fluorescent hybridization across young, middle age and old Fisher Brown Norway rats (an aging model that acquires low frequency presbycusis) we quantified individual GAD1 mRNA in small, medium and large GABAergic cells. Our results demonstrate that small GABAergic cells in low frequency regions had ~58% less GAD1 in middle age and continued to decline into old age. In contrast, the amount of GAD1 mRNA in large cells in low frequency regions significantly increased with age. As several studies have shown that downregulation of GAD1 decreases the release of GABA, we interpret our results in two ways. First, the onset of presbycusis may be driven by small GABAergic cells downregulating GAD1. Second, as previous studies demonstrate that GAD67 expression is broadly downregulated in the old IC, perhaps the translation of GAD1 to GAD67 is interrupted in large GABAergic IC cells during aging. These results point to a potential genetic mechanism explaining reduced temporal precision in the aging IC, and in turn, presbycusis.

Tonotopic distribution and inferior colliculus projection pattern of inhibitory and excitatory cell types in the lateral superior olive of mice.

The principal neurons (PNs) of the lateral superior olive nucleus (LSO) are an important component of mammalian brainstem circuits that compare activity between the two ears and extract intensity and timing differences used for sound localization. There are two LSO PN transmitter types, glycinergic and glutamatergic, which also have different ascending projection patterns to the inferior colliculus (IC). Glycinergic LSO PNs project ipsilaterally while glutamatergic one's projections vary in laterality by species. In animals with good low-frequency hearing (<3 kHz) such as cats and gerbils, glutamatergic LSO PNs have both ipsilateral and contralateral projections; however, rats that lack this ability only have the contralateral pathway. Additionally, in gerbils, the glutamatergic ipsilateral projecting LSO PNs are biased to the low-frequency limb of the LSO suggesting this pathway may be an adaptation for low-frequency hearing. To further test this premise, we examined the distribution and IC projection pattern of LSO PNs in another high-frequency specialized species using mice by combining in situ hybridization and retrograde tracer injections. We observed no overlap between glycinergic and glutamatergic LSO PNs confirming they are distinct cell populations in mice as well. We found that mice also lack the ipsilateral glutamatergic projection from LSO to IC and that their LSO PN types do not exhibit pronounced tonotopic biases. These data provide insights into the cellular organization of the superior olivary complex and its output to higher processing centers that may underlie functional segregation of information.

Age-related ultrastructural changes in the lateral cortex of the inferior colliculus.

Temporal precision, a key component of sound and speech processing in the inferior colliculus (IC), depends on a balance of inhibition and excitation, and this balance degrades during aging. The cause of disrupted excitatory-inhibitory balance in aging is unknown, however changes at the synapse are a likely candidate. We sought to determine whether synaptic changes occur in the lateral cortex of the IC (IClc), a multimodal nucleus that processes lemniscal, intrinsic, somatosensory, and descending auditory input. Using electron microscopic techniques across young, middle age and old Fisher Brown Norway rats, our results demonstrate minimal loss of synapses in middle age, but significant (∼28%) loss during old age. However, in middle age, targeting of GABAergic dendrites by GABAergic synapses is increased and the active zones of excitatory synapses (that predominantly target GABA-negative dendrites) are lengthened. These synaptic changes likely result in a net increase of excitation in the IClc during middle age. Thus, disruption of excitatory-inhibitory balance in the aging IClc may be due to synaptic changes that begin in middle age.

Tonotopic distribution and inferior colliculus projection pattern of inhibitory and excitatory cell types in the lateral superior olive of Mongolian gerbils.

Sound localization critically relies on brainstem neurons that compare information from the two ears. The conventional role of the lateral superior olive (LSO) is extraction of intensity differences; however, it is increasingly clear that relative timing, especially of transients, is also an important function. Cellular diversity within the LSO that is not well understood may underlie its multiple roles. There are glycinergic inhibitory and glutamatergic excitatory principal neurons in the LSO, however, there is some disagreement regarding their relative distribution and projection pattern. Here we employ in situ hybridization to definitively identify transmitter types combined with retrograde labeling of projections to the inferior colliculus (IC) to address these questions. Excitatory LSO neurons were more numerous (76%) than inhibitory ones. A smaller proportion of inhibitory neurons were IC-projecting (45% vs. 64% for excitatory) suggesting that inhibitory LSO neurons may have more projections to other regions such the lateral lemniscus or more distributed IC projections. Inhibitory LSO neurons almost exclusively projected ipsilaterally making up a sizeable proportion (41%) of the transmitter type-labeled ipsilateral IC projection from LSO and exhibited a moderate low frequency bias (10% difference H-L). Two thirds of excitatory neurons projected contralaterally and had a slight high frequency bias (4%). One third of excitatory LSO neurons projected ipsilaterally to the IC and these cells were strongly biased toward the low frequency limb of the LSO (37%). This projection appears to be species specific in animals with good low frequency hearing suggesting that it may be a specialization for such ability.