The GABA Responses In The Rat Mesencephalic Trigeminal Neurons And Associated Developmental Studies

It is well known that GABA is the major inhibitory neurotransmitter in the central nervous system of the mammalian animals and plays important roles in many neuronal processes through binding with corresponding receptors. Previous studies have showed that GABAergic axonal terminals surround the soma of the mesencephalic trigeminal nucleus(Vme) and our research group has proved that GABAergic axonal terminals make synapses with the soma of the Vme at the ultrastructure level. At the same time, the expression of GABAARα1 and GABAARα3 was also observed. Some reports showed that the responses of Vme to GABA were excitatory. However, due to the limitation of the methods, it is still unclear about the exact GABA responses in rat Vme and the possible molecular basis of the process. Cation-Cl- cotransporters, which were responsible for the transportation of chloride inside and outside of the cell, were supposed to be involved in the GABA responses. Until now, no direct evidence has been reported. Cation-Cl- cotransporters have been considered to play key roles in controlling the intracellular and extracellular ionic environments of neurons. To date, seven members of the cation-Cl- cotransporter gene family have been reported. In the nervous system, KCC1, KCC2, KCC3 and NKCC1 have been found to be expressed. Among these, KCC2 mainly extrude Cl- out of the cell, in contrast, NKCC1 promotes accumulation of Cl- in the cell. The stability and balance of [Cl-]i depond on the expression of KCC2 and NKCC1 on the membrane of the neuron. But the change of expression of KCC2 and NKCC1 during the postnatal stage in the rat Vme has not been reported and the change may be associated tightly with the final effect of opening of the chloride channel.Parvalbumin (PV) is an important member of the calcium binding proteins. Previous developmental studies have showed that PV is specially distributed in the soma and process of the primary proprioceptive neurons such as large DRG neurons and Vme neurons. Therefore PV has been regarded as an excellent marker for primary proprioceptive neuronal populations. On the other hand, the developmental studies on the Vme have reported that PV is widely distributed in all Vme neurons during the whole developmental stage. During the postnatal stage, whether PV and KCC2 or NKCC1 colocalized in the Vme and whether the percentage of colocalization varied is not clear and whether the variation coincided with the forementioned results is also not clear.To answer these questions, in the present study, an attempt was made to examine: (1) the GABA responses in mechanically dissociated Vme neurons with attached synaptic boutons by using the whole cell patch clamp technique; (2) the change of expression of KCC2 and NKCC1 during the postnatal stage in the Vme by using the Western blotting technique; (3) the percentage of colocalization of PV with KCC2 or NKCC1 during the postnatal stage in the Vme by using the double immunofluorescence histochemical techniques. Part One The GABA responses in the rat Vme neuronsIn this study, the whole cell patch clamp technique was used to investigate the GABA responses in the mechanically dissociated Vme neurons. The results are as follows: inward mPSCs were produced at a holding potential of -70 mV in the presence of 1μM TTX, 10μM CNQX and 10μM AP-5, the average amplitude of mPSCs was 24±20 pA,and the reversed potential was about -30 mV which was close to the Cl- equilibrium potential calculated by Nernst equation. The mPSCs induced by GABA released from the presynaptic boutons could be completely and reversibly blocked by 5μM bicuculline, which is the specific antagonist of GABAA receptor.These results indicate that GABA released from the presynaptic boutons may produce modulatory effects through binding with GABAA receptor on postsynaptic Vme neurons. The activation of GABAA receptors result in the opening of the chloride channel, the flow of Cl- between the inside and outside of the membrane of Vme neurons may be the basis of GABA's effect on Vme neurons.Part Two The change of expression of NKCC1 and KCC2 in the rat Vme neurons during the postnatal stageIn this study, we selected six postnatal time points: P0, P4, P7, P10, P14 and P21 to examine the change of expression of KCC2 and NKCC1 in the rat Vme by using the Western blotting combined with image analysis. The results are showed: (1) NKCC1 is expressed only in the soma of the Vme, not in the axons or dendrites; (2) NKCC1 has been expressed weakly at P0 in the Vme neurons, with the development, the expression of NKCC1 increased gradually, and the expression level of NKCC1 reached the adult level at P10, and kept stable until the adult time. Gray scale analysis with Image-pro plus software showed that the value of gray scale increased from P0 to P21, and significant differences existed between P0, P4, P7 and P10, P14, P21 when the value of gray scale of the P0, P4 or P7 was compared with the value of gray scale of the P10, P14 or P21, respectively (P < 0.05). However, the expression of KCC2 kept weak at all the time points and no change was observed between time points (P > 0.05).The above results suggest that the increasing expression of NKCC1 and the weak expression of KCC2 during the postnatal stage of the rat Vme neurons may be the major cause of high level of [Cl-]i in adult Vme neurons, which is closely correlated with the GABA responses in the Vme neurons.Part Three The colocalization study of PV with NKCC1 or KCC2 in the rat Vme during the postnatal stageIn this part, we selected five time points: P4, P7, P10, P14 and P21 to investigate the colocalization of PV with NKCC1 or KCC2 during the developmental stage by using the double immunofluorescence histochemical technique. The results showed that with the development the percentage of the colocalization of PV with NKCC1 increased, however, the expression of KCC2 could not be detected. Whereas, the colocalization of PV with KCC2 could be detected in the Purkinje cell of the cerebellum as for the positive control experiments. These results further indicate that the high expression of NKCC1 and the weak expression of KCC2 in the adult Vme neurons may be the molecular basis of the GABA responses in the adult Vme neurons.