Circadian Modulation Of GABA On The SEPSC Of The Lateral Habenular Nucleus

Object: To characterize the synaptic currents induced by GABA in themedial and lateral habenular neurons from different phases and to determinewhich GABA receptor subtypes mediate these effects.Methods: Whole cell voltageclamprecordings were made from slices of thehabenular nucleus (Hb) in 130150grats. To investigate the response toγaminobutyric acid (GABA) in medial and lateral habenular neurons fromdifferent phases (at CT 1018and CT 204for day and night experiments,respectively), GABA and bicuculline (Bic, the GABAA receptor antagonist)were applied to brain slices by bath perfusion. Recordings were made at 30℃and at a holding potential of 60mV. Data were lowpassfiltered at 1 kHzand sampled at 100μs intervals. Experimental data were recorded using apersonal computer running Clampex 9 (Axon Instruments) connected to aAxopatch 200B amplifier (Axon Instruments) via a Digidata 1322A digitizer(Axon Instruments). Data were analyzed using the Clampfit 9 and Originsoftware packages.Result: 1.Under basal conditions,the sEPSC frequency in LHb neuronsshowed a significant daynightdifference with high during the day(2.23±0.63Hz)and low during the night(0.75±0.22Hz). But under basalconditions, the sEPSC frequency in MHb neurons did not have a significantdifferent between the day and the night. 2.The effect of GABA on the sEPSC inLHb neurons was different between the day and the night. During the day ,GABA induced 44±8% decreased in sEPSC frequency of LHb neurons. Duringthe night, GABA induced a significantly increased in sEPSC frequency (49±7%)in 61% of LHb neurons. These GABAinducedchanges in sEPSC frequency were blocked by bicuculline the GABAA receptor antagonist. 3.GABA resultedin a reduction in the sEPSC frequency of MHb neurons during both day(29±6%) and night (23±6%). Moreover, GABA induced decrease in sEPSCfrequency of MHb neurons were not significant differerce between the day andthe night. This GABA induced decrease in sEPSC frequency was not blockedby bicuculline.Discussion: 1. Excitatory effects of GABA on the sEPSC in LHb neuronsduring the night.The present results showed that the effect of GABA in thesEPSC of LHb neurons exhibited a circadian rhythm. GABA induced asignificant increase in sEPSC frequency in 61% of LHb neurons during thenight and decrease during the day. The effects of GABA were variable andmean change was not significant, whereas GABA induced changes in sEPSCfrequency were significantly different between day and night. These GABAinduced change in sEPSC frequency were blocked by bicuculline the GABAAreceptor antagonist, which indicated that these changes were mediated byGABAA receptors. GABA resulted in a reduction in the sEPSC frequency inMHb neurons during both day and night. Moreover, this GABA induceddecrease in sEPSC frequency was not blocked by bicuculline. These resultswere in agreement with absence of GABAAR in adult MHb neurons. Thisindicated that the increase in sEPSC frequency of LHb neurons caused adepolarization of membrane potential during the night, resulting in a decrease inthe firing threshold, and consequently a increase in the probability of actionpotential initiation, causing neuronal excitation. Is opposite with it during theday. Thus the GABAergic neurons may passes excitatory inputs during thenight but cuts off them during the day. What factors or mechanisms determine the excitatory effect of GABA on the sEPSC in LHb neurons during the night?The most conspicuous electrical property common to all lateral habenular cellswas the production of a rebound,burst discharge of action potentials in responseto a brief hyperpolarizing current pulse.Lateral habenula neurons were found tohave two distinct modes of activity: 1) tonic firing and 2) burst firing.Atmembrane potentials more positive than 65mV, depolarization elicited trainsof sodiumdependentfast action potentials. At membrane potentials morenegative than 65mV,slight depolarization elicited a TTX insensitive wave ofdepolarization, called a lowthresholdspike (LTS), from which a burst of fastaction potentials were triggered. LTS was related to a lowthresholdcalciumchannels, which is inactivated at membrane potential more positive than 65mV and deinactivated when the membrane is hyperpolarized to potentials morenegative than 65V.The major population of neurons in this area generatesprolonged discharges of action potentials in response to a brief membranehyperpolarization.Following short hyperpolarizing current pulses, reboundLTSs often occur in a series, at frequencies ranging from 3 to 10 Hz. Theseelectrophysiological properties of neurons in the LHb nucleus indicated thatthere was a GABAergic network in the LHb nucleus.Thus the GABAergicnetwork may behave as an interface that converts inhibitory influences of thepresynaptic neurons into excitatory ones and conveys these excitatoryinfluences to the postsynaptic neurons through the rebound activity. Moreover,these rebound activity occur with approximately the same frequency (310Hz)as synchronized populations of LHb neurons. Therefore, the LHb can integrateinformation received from presynaptic neurons and subsequently modulateactivities of the postsynaptic neurons. On the other hand, K + /Cl cotransporter, KCC2 might be key factor for GABAA–mediated excitatory effects during thenight. GABA was the main inhibitory transmitter in the adult brain. However,during early neuronal development, GABAAreceptormediatedresponses areoften depolarized. The development led to change in GABAA mediatedresponses from depolarizing to hyperpolarizing. GABA mediated synapticinhibition by opening postsynaptic ligandgatedCl－channels. Their openingalways led to an outward current and to a hyperpolarization of the postsynapticmembrane that can be inhibitory. Low intracellular chloride concentrations([Cl－]i) in postsynaptic neurons was required for GABAARmediatedinhibitory.KCC2 expression was important to an efficient chloride extrusion thatmaintained the Cl－equilibrium potential of mature neurons at values morenegative than the resting membrane potential. Several experimentsdemonstrated a tight link between the expression of KCC2 and GABAAmediatedhyperpolarizing responses. Previous studies have implicated that theneurons of olfactory bulb, SCN and the MHb lack KCC2 mRNA. Olfactorybulb and SCN nucleus neurons show excitatory responses to GABA. A largesubgroup of SCN neurons had markedly depolarized GABAA reversalpotentials in the night phase.It is tempting to speculate that the expression ofKCC2 may differert during day and night.The upregulation of KCC2expression during the day may determine GABA mediated inhibitory effect.The downregulation of KCC2 expression during the night may determine theswitch of GABA from excitatory to inhibitory effect. 2.Inhibitory effects ofGABA on the sEPSC of LHb neurons during the night. During the night, theeffects of GABA on the sEPSC of LHb neurons were variable, and the meanchange was not significant. The main reason was that GABA induced a decreased in sEPSC frequency in 39% of LHb neurons during the night. Eventoday, there was lack of elaborate subnucleus localization criteria dependenton neurophysiological properties, its neuronal circuits and neurotransmitter,which may provide the physiological and molecular basis for its diversebiological functions. During the night , LHb neurons have different effectsresponses to GABA may due to they were belong to the different cellularsubsets. Moreover, application of exogenous GABA may cause massiveshunting that is the same as shunting inhibition during longlastingapplicationof excitatory neurotransmitters like glutamate. Such a shunting effect caused bythe massive opening of GABAA receptor Cl－channels, resulting in a stronglyincreased conductance and accompanying depolarizing GABA current. Thisresults in an effective"clamping"of the membrane at the Cl－reversal potential.Such depolarizing shunting will curtail the ensuing spike repolarization,preventing Na+ channels from becoming deinactivated. 3. What is the role ofLHb in the circadian timing system. Formerly studied showed that LHb andSCN had many similarities characteristics.For example, almost half of theLHbM cells were photically responsive and VL part of SCN received opticalinputs from retina via a direct retinohypothalamic tract. Moreover, the SCN andLHb also share the characteristic that photic responses are larger at night thanduring the day. Both Hb and SCN exhibited a circadian rhythm of baselinefiring rate, with higher discharge activity during the day and lower during thenight. GABAA receptors were abundantly expressed in LHb and SCN neurons.There is considerable evidence indicating that GABA plays a central role incircadian timekeeping.The LHb appears to be innervated by the SCN, asdescribed in the golden hamster using anterograde tracing with Phaseolus vulgaris leucoagglutinin. The SCN and LHb contained copiously melatoninreceptors.Melatonin plays a role in the regulation of circadian rhythmicity andsleep. Older anatomical and electrophysiological data have suggested theexistence of a direct habenulapinealprojection, and pinealocyte projections tothe habenula. The vasopressinergic fibers originated from the SCN are presentin the medial division of the lateral habenula in the rat. The vasopressinergicneurons were pacemaker neurones in the SCN.This research manifested thatThe effects of GABA on the sEPSC of LHb neurons exhibited a circadianrhythmicity, inhibitory effects during the day and excitatory effects during thenight. This effects of GABA on the LHb were affinis on the SCN. In addition,inagreement with our previous studies ,under basal conditions sEPSC frequencywere high during the day and low during the night. These indicated thathabenular nucleus may play a considerable role in the circadian timing system.How LHb might be involved in such effects requires further investigation.Andwhat the mechanisms might be that GABA mediated excitatory effects on thesEPSC in LHb neurons during the night requires futher reseach.Conclusion: 1. LHb neurons exhibited a circadian rhythmicity in sEPSCfrequency, high during the day and low during the night. MHb neurons did notexhibit a circadian rhythmicity in sEPSC frequency.2. The effects of GABA onthe sEPSC of LHb neurons revealed a significant daynightdifference withinhibitory effects during the day and excitatory effects during the night.3. Theeffects of GABA on the sEPSC in LHb neurons were mediated by GABAAreceptors.