Font Size: a A A

Peri-adolescent Alteration Of Tonic Inhibitory Current In Lateral Amygdala

Posted on:2014-11-03Degree:DoctorType:Dissertation
Country:ChinaCandidate:P WeiFull Text:PDF
GTID:1264330425450528Subject:Neurobiology
Abstract/Summary:PDF Full Text Request
Adolescence is a human’s transitional duration of physical and psychological development, which generally occurs during the time from puberty to adult. The adolescent period always occurs close to the teenage years and the brain appeared changes that have substantial implications for behavior during this period. In fact, the brain has reaches90%of adult volume by six years old. So, the brain does not increase in volume much during adolescent period. The biggest changes in the brain during this period emerge in the cortex that deals with cognitive and emotional information.During adolescent period, people were reported to have some different behavior from adult, including risk-taking, easily irritating, emotional violence and so on. Accumulating evidences demonstrated that the specific behavior during this period was closely related to the amygdala, which is main region processing emotion in the brain. Not so long ago amygdala was an obscure region of the brain that attracted relatively little scientific interest, but today it is one of the most heavily studied brain areas, and practically a household word. The amygdala was first recognized as a distinct brain region in the early19th century. The name, derived from the Greek, was meant to denote an almond-like shape structure in the medial temporal lobe.Accumulating evidence indicated that local inhibitory circuits in the amygdala contribute to important aspects of fear conditioning and extinction. Systemic or local treatments that increase GABAergic transmission produce anxiolytic effects and can interfere with the acquisition or expression of conditioned fear responses. In contrast, pharmacological manipulations that decrease GABAergic transmission induce anxiogenic-like effects and can improve learning or retrieval of conditioned fear.The inhibitory transmitters GABA exert its function through two different ways. One way is that GABA is released from presynaptic terminal and bound to the postsynaptic receptor in the cleft. This classical way is a point-to-point mode, called phasic inhibition. In the other form, the GABA spill out of the synaptic cleft and bound to the extrasynaptic receptors. This type of receptor has a high affinity with GABA and has a slow desensitization. So the GABA receptor could persist to be active, we call it tonic inhibition.Tonic inhibition persistently induces the charge to flow from one side o the membrane to the other to modulate the excitability of the neurons. It leads to a3.5fold anion charge across membrane as the phasic inhibition. In addition to this function, a bunch of evidences showed that tonic GABAA receptor currents are also expressed in neuronal progenitor cells, indicating they might play a part in neural development. In cerebellar granule cells, the tonic GABAA-receptor-mediated conductance increases with maturation, while IPSCs become smaller and faster. The tonic conductance becomes comparable to the peak amplitude of the phasic component in granule cells in the adult. In another research, hippocampal DG granule cells appear to show evidence of a robust tonic GABAA-receptor-mediated current during the first two postnatal weeks and decreased significantly in adult. And in VBN, neurons showed the remarkable change of tonic inhibition current during development.Whether tonic inhibition current also exists in the amygdala has not been investigated until a recent research reports. They found out that principal cells in the lateral amygdala could express the tonic inhibition currents, but little is known about the developmental change of tonic inhibition current in amygdala. Given that the important role of tonic inhibition current in the brain development, we decided to investigate the development change of tonic inhibition in the lateral amygdala principal cells.Firstly, we recorded and calculated tonic inhibition current by subtracting the I-hold current of20uM GABA from that of BIC and demonstrated the result that amygdala exhibits different tonic GABAergic current at postnatal20,30-40and60days. LA principle neurons at P30-40exhibited weaker tonic inhibition current amplitude (10.56±1.11pA, n=14) than that at P15-20(42.05±6.75pA, n=20) and P55-60(29.79±4.59pA, n=10). And there no significant difference between the groups of P15-20and P55-60(P>0.05).To further determine specificity of this change in the LA PCs, we investigate whether tonic inhibition currents were similar subject to developmental changes in hippocampus CA1and mPFC region. We used the same concentration of GABA as in the amygdala to induce tonic currents and compared its magnitude at P15-20, P30-40and P55-60. At postnatal15-20days, hippocampus CA1pyramidal neurons (PNs) produced an tonic inhibition current of43.99±11.83pA (n=7). A similar response was recorded in P30-40hippocampus CA1PNs (38.83±11.75pA; n=7). The tonic current in P55-60hippocampus CA1PNs was48.13±15.51pA, not significantly different from that of P15-20and P30-40hippocampus CA1PNs (n=7, P>0.05; ANOVA). We therefore demonstrated that the tonic inhibition current amplitude in hippocampus CA1PNs exhibited no significant changes across the three developmental stages. As for the prefrontal cortex, the tonic currents were31.70±4.99pA,25.75±6.86pA and24.98±3.70pA at P15-20, P30-40and at P55-60respectively in the pyramidal neurons. The magnitude of the current also presented no significant changes (n=6; P>0.05, ANOVA) along with development. These results indicated that tonic inhibition has less modulation on the amygdala than in the hippocampus and mPFC during adolescence period (P30-40), and due to this, amygdala is more active in the adolescence and it is difficult for youngsters to control their emotion.Next, we want to know which type subunit of the GABAA receptor mediated this tonic inhibition current. We used the neuropharmacological method (agonists or antagonists to different subunits of GABAA receptor) to investigate the tonic inhibition current in LA PCs of P15-20mice. To assess the component mediated by a5-GABAARs in these cells, we measured tonic inhibition after application of L-655,708, the α5-GABAARs inverse agonist selectively. The results showed that L-655708did not significantly decrease the responses caused by the GABA, but bicuculin (10μm) completely inhibited the tonic GABA-mediated currents in LA PCs (L655708-GABA:1.29±1.82pA, n=6, P>0.05). This demonstrated that tonic inhibition current was not caused by a5-GABAARs. In another experiment, we utilized THIP, we use a specific agonist of δ-GABAA receptors to the bath ACSF instead of application of GABA. Five LA PCs showed a small amplitude of4.34±0.90pA, indicating that δ subunit was not the component of the tonic GABAA receptors in the LA PCs, at least not the main subunit. This indicated that a totally distinct subunit might mediate the tonic inhibition current in amygdala and emerge as a wholly new subunit, which could induce tonic inhibition.To further characterize tonic currents in the LA PCs, we applied the α3GABAAR selective benzodiazepine agonist TP003. The drug significantly induced tonic inhibition currents in LA PC at different time points (P15-20:36.07±6.79pA; P30-40:10.42±1.94pA; P55-60:26.59±4.59pA). The amplitude at P30-40was significantly smaller than that in groups of P15-20and P55-60, but there was no difference between other two groups (n=6; P<0.01;ANOVA). In addition, TP003had no significant effect on sIPSC amplitude and frequency, which confirms the negligible role for α3GABAARs in synaptic transmission. To verify the contribution of α3GABAARs to tonic inhibitory currents, we further compared the difference between tonic currents caused by TP003and20μM GABA at P15-20period. Results showed that20μM GABA-induced magnitude of tonic currents in these cells was stronger than that observed with TP003alone, but without significant difference [GABA:42.05±6.75pA (n=20) vs TP003:36.07±6.79pA (n=6), P>0.05; unpaired Student’s t test]..We next wanted to study whether tonic inhibitory currents in LA PCs are sufficiently large to affect the excitability of these cells. In order to avoid the influence of the glutamatergic synapse activity, we performed the experiments to induce action potentials via somatic current injections under complete block of ionotropic glutamatergic transmission (2mM kynurenate). Results showed that during the adolescent period (P30-40), TP003had no significant effect on the numbers of action potential in the LA PCs (numbers of AP:P30-40con5.6±0.19spikes vs TP0035.72±0.26spikes vs BIC5.48±0.25spikes; n=5,P>0.05; ANOVA). In the pre-adolescence (P15-20) and early adult (P55-60) mice, application of TP003significantly reduced the numbers of AP spikes (numbers of AP:P15-20con6.56±0.17spikes vs TP0033.04±0.12spikes vs BIC6.44±0.17spikes; P55-60con6.32±0.17spikes vs TP0034.08±0.16spikes vs BIC6.52±0.14spikes; n=5, P<0.01; ANOVA). Results also presented that application of BIC significantly increased the number of APs, which was suppressed by TP003. These results demonstrated that a3GABAA receptors might differently express and modulate the excitability of LA PCs during development.Then we investigated the mechanism by which a3subunit modulate the activation of principal cells. We examine whether membrane potential was affected by the α3subunit in the LA PCs. The resting membrane potential of principal cells in the lateral amygdala was estimated from the reversal potential of voltage-gated K+currents activated by command ramp potentials applied via a cell-attached pipette. The results showed that the resting potential of neurons was not significantly changed after the TP003administration, compared to the control (resting potential:con-70.55±2.01mV vs TP003-70.10±2.88mV, n=8, P>0.05).In conclusion, our results here showed the tonic inhibitory currents induced by α3GABAA receptor were changed during adolescent period in mice LA PCs and α3GABAA receptor could exert its function by suppressing the excitatory action of LA PCs. This mechanism might explain the animal is inclined to be affected by emotional events in adolescent period.
Keywords/Search Tags:tonic inhibitory current, α3subunit, amygdala, lateral amygdala, GABAergic inhibition
PDF Full Text Request
Related items