Deafness disrupts chloride transporter function and inhibitory synaptic transmission

Abstract

Loss of sensory function leads to atrophy or death within the developing CNS, yet little is known about the physiology of remaining synapses. After bilateral deafening, gramicidin-perforated-patch recordings were obtained from gerbil inferior colliculus neurons in a brain slice preparation. Afferent-evoked IPSPs had a diminished ability to block current-evoked action potentials in deafened neurons. This change could be attributed, in part, to a loss of potassium-dependent chloride transport function, with little change in K-Cl cotransporter expression. Treatments that suppressed chloride cotransport (bumetanide, cesium, and genistein) had little or no effect on neurons from deafened animals. These same treatments depolarized the E(IPSC) of control neurons. Semiquantitative RT-PCR and immunohistochemical staining indicated no change in the expression of chloride cotransporter mRNA or protein after deafness. Therefore, profound hearing loss leads rapidly to the disruption of chloride homeostasis, which is likely attributable to the dysfunction of the potassium-dependent chloride cotransport mechanism, rather than a downregulation of its expression. This results in inhibitory synapses that are less able to block excitatory events.

Figure 1.

An evaluation of inhibitory synaptic strength in neurons from control and BCA animals. A, Traces show examples of LL-evoked IPSPs, current-evoked action potentials, and the simultaneous presentation of IPSPs with action potentials (from left to right) in control and BCA animals. The number of times that the LL-evoked IPSP inhibited the AP in 10 consecutive trials was counted and used to calculate the percentage of inhibition. B, Mean IPSP ability to inhibit APs is significantly lower in BCA neurons compared with controls. n values are in bars (^*p < 0.0001). C, Duration of inhibition was significantly shorter in BCA neurons compared with controls (measurement described in Materials and Methods). n values are in bars (^**p < 0.005). Error bars indicate SEM.

Figure 2.

The effect of K⁺ or Cs⁺ in the recording pipette on E_IPSC in neurons from control and BCA animals. A, Representative traces from control and BCA animals at different holding potentials are shown for recordings with K⁺- and Cs⁺-containing pipettes. B, For control neurons, the mean E_IPSC was significantly more depolarized when the internal pipette solution contained Cs⁺. However, for BCA neurons, there was not a significant difference between K⁺- and Cs⁺-containing pipettes. n values are in bars (^*p < 0.0001 vs K⁺). Error bars indicate SEM.

Figure 3.

The effect of genistein on E_IPSC in neurons from control and BCA animals. A, Representative traces from control and BCA animals at different holding potentials are shown for recordings obtained in ACSF and after exposure to 50 μm genistein. KYN was present throughout these recordings. B, The mean E_IPSC for control neurons was significantly depolarized in the presence of genistein. However, for BCA neurons, there was not a significant difference. n values are in bars (^*p = 0.005 vs E_IPSC in ACSF). Error bars indicate SEM.

Figure 4.

Chloride cotransporter mRNA expression in the ICs from control and BCA animals. A, Amplification of NKCC1, KCC2, and GAPDH sequences in gerbil and rat yielded bands of identical size. Primers used were specific for either NKCC1 (top) or KCC2 (bottom), and two different primer sets were used for each of the genes (described in Materials and Methods). GAPDH mRNA was amplified from the same samples as an input control. The number of PCR cycles was 30. B, NKCC1 mRNA expression in control (Con) and BCA gerbils. RNA was isolated from gerbils at days 9 (P9) and 14 (P14) and was subjected to RT-PCR. Sizes of amplified DNA fragments are indicated on the left, and the number of PCR cycles is indicated on the right. C, KCC2 mRNA expression in control and BCA gerbils. RNA was isolated from gerbils at P9 and P14 and was subjected to RT-PCR. Sizes of amplified DNA fragments are indicated on the left, and number of PCR cycles is indicated on the right.

Figure 5.

Immunohistochemical staining with anti-KCC2 polyclonal antibody in the ICs of control and BCA animals. Top, Control section in which tissue was not exposed to primary antibody. Middle, KCC2 staining in the IC of control animal. Bottom, KCC2 staining in the IC of BCA animal. Images were obtained at a magnification of 630×. Scale bar, 50 μm.