Mechanisms Underlying The Raised Activity Of Serotonergic Neural Circuits In Rodent Dorsal Raphe Nucleus By Sodium Salicylate

BACKGROUND:Serotonin or5-hydroxytryptamine (5-HT) is a monoamine neurotransmitter or neuromodulator which is primarily found in the gastrointestinal tract and the central nervous systems (CNS) of animals and humans. In the CNS, the cell bodies of serotonergic (5-HTergic) neurons are distributed in the raphe nucleus and provide axonal projections to various brain areas, including the auditory system. The5-HTergic system has been implicated in many neurological symptoms, including tinnitus, a phantom auditory sensation in the absence of an external sound. Tinnitus can be caused by a variety of factors, including noise exposure or ototoxic drugs. It has been reported that sodium salicylate (NaSal), the main metabolite of aspirin (acetylsalicylic acid) that can induce tinnitus, can significantly increase extracellular5-HT levels in the inferior colliculus and the auditory cortex. It is also reported that the activity of neurons, including5-HTergic neurons, are enhanced significantly in the dorsal raphe nucleus (DRN) of gerbils with NaSal-induced tinnitus. The present study aimed to explore mechanisms underlying the enhanced5-HTergic neural activity by NaSal. Specifically, I examined effects of NaSal on functional intrinsic properties and synaptic transmissions of5-HTergic and GABAergic neurons in slices of DRN in rats and mice.METHODS:Brain slices were prepared from the Wistar rat and the VGAT-ChR2-EYFP transgenetic mouse. In the transgenic mouse, light-sensitive channelorhodopsin-2is expressed in the GABAergic neurons. Whole-cell patch-clamp technique was used to record the intrinsic memebrane properties and the synaptic transmissions of5-HTergic and GABAergic neuruns in the DRN. Specifically, the membrane input resistance, membrane potential, spontaneous firing, current or light-evoked firing, and the spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) and excitatory postsynaotic currents (sEPSCs and mEPSCs) were recorded. Brain slices were perfused with1.4mM NaSal which changes in the intrinsic memebrane properties and the synaptic transmissions were recorded.In the Wistar rat,5-HTergic neurons and GABAergic neurons were identified by the morphological, electrophsiological and pharmacological properties. In the VGAT-ChR2-EYFP transgenetice mouse,5-HTergic and GABAergic neurons were identified by the morphological, electrophsiological, pharmacological and optogenetic properties.RESULTS:In DRN slices of rats,(1) the5-HTergic neurons and GABAergic neurons were identified successfully through their distinct morphological, electrophysiological and pharmacological response properties.(2) For GABAergic neurons, NaSal reversibly hyperpolarized the resting membrane potential, decreased the membrane input resistance, changed the action potential properties and depressed the spontaneous and current-evoked firing, but not affected those in5-HTergic neurons. These results indicate that NaSal preferentially alters the intrinsic membrane properties and suppresses the membrane excitability in GABAergic neurons.(3) NaSal suppressed the frequency and amplitude of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in5-HTergic and GABAergic neurons, and reduced the frequency and amplitude of miniature IPSCs (mIPSCs) in GABAergic neurons, but only suppressed the frequency, not the amplitude, of mIPSCs in5-HTergic neurons. These results suggest that NaSal reduces the GABAergic synaptic transmission in GABAergic neurons through both presynaptic and postsynaptic mechanisms, and suppressed that in5-HTergic neurons mainly through a presynaptic mechanism.(4) NaSal increased the frequency, not the amplitude, of glutamatergic sEPSCs and mEPSCs in5-HTergic neurons, but not in GABAergic neurons. This result indicates that NaSal enhances the efficiency of glutamatergic synaptic transmissi in5-HTergic neurons through presynaptic mechanism in rat DRN.In DRN slices of the VGAT-ChR2-EYFP transgenetic mouse,(5) a blue laser light (473nm) could excite the GABAergic neurons, and strongly hyperpolarized the membrane potential and eliminated current-evoked firing in5-HTergic neurons. This result indicates that optical stimulation could enhance the inhibitory level in the transgenic mouse DRN through exicting GABAergic neurons.(6) NaSal significantly suppressed the light-evoked firing in GABAergic neurons. During the blue light stimulation, NaSal significantly increased the current-evoked firing, increased the membrane input resistance and depolarized the resting membrane potential in5-HTergic neurons. These results indicate that NaSal could disinhitbit5-HTergic neural circuits in the DRN slices.(7) As it did in rat DRN, NaSal suppressed blue light stimulation-induced enhancement of the sIPSC activity in5-HTergic neurons, indicating that NaSal reduces the inhibitory synaptic transmissions to5-HTergic neurons in the transgenetic mouse DRN. DISCUSSION:In the present study, the results indicate that NaSal has direct effects on the neuronal excitability and synaptic transmissions of the rodent DRN. NaSal could promote the neural activity of local DRN5-HTergic neural circuits by suppressing GABAergic activities and by enhancing the glutamatergic synaptic transmission to5-HTergic neurons in the slices. Evidence supporting this conclusion includes:(1) NaSal increased neuronal excitability of5-HTergic neurons, but decreased that of GABAergic neurons;(2) NaSal reduced inhibitory synaptic transmission to5-HTergic neurons;(3) NaSal enhanced glutamatergic synaptic transmissions to5-HTergic neurons. Enhanced activity of5-HTergic neural circuits in the DRN by NaSal may serve as a neural basis for the boosting action of NaSal on5-HT level in the brain.