Group+1

** Professor: Philip Chu ** ** Authors: Eva David, Theologia Karagiorgis, Sarah Leibowitz ** ** Published: December 17, 2012 **
 * Final Project- Experimental Psychology: Neurobiology **

** The Excitatory Effects of GABA and its Transformation to an Inhibitory Neurotransmitter **

** Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter used in adult central nervous system synapses. It is produced from glutamate via glutamic acid decarboxylase (GAD) and is distributed across a large number of synapses in the mammalian brain (Bear et al., 2007). GABA generally acts to inhibit neuronal activity by causing inhibitory post-synaptic potentials (IPSPs) that hyperpolarize the post-synaptic membrane, or by shunting excitatory inputs before they reach the action potential trigger zone. GABA-gated channels mediate the flow of Cl- ions across the membrane. At rest, there are relatively set concentration gradients for chloride – there is a greater extracellular chloride concentration as compared with the intracellular environment for a mature neuron. This concentration difference contributes to a negative equilibrium potential for chloride. Thus, when GABA binds its respective receptor, Cl- ions diffuse down their concentration gradients and into the cell, driving the membrane potential to a more negative value, inhibiting its ability to reach the threshold voltage necessary to fire an action potential. It is important to note that the inhibition implemented in the post-synaptic cell is not a direct result of GABA release from the pre-synaptic cell, but is due rather to the electrochemical chloride gradient. As we will see, GABA can induce depolarization and cause action potentials under specific conditions. In this review, we will be examining how variable intracellular chloride concentrations, differential expression of Cl- transporters and increased energy supply contribute to conversion of a GABAergic synapse from excitatory to inhibitory. ** **  The inhibitory role of GABA in the nervous system has been studied in mature neurons, however research on developing, immature neurons has shown that GABA may mediate excitatory transmission as well. Ben-Ari et al. (1989) studied the effects of intracellular Cl- ion concentration differences in cultured rat hippocampal neurons during the first two weeks of post-natal development. Giant depolarizing potentials (GDPs) were recorded from hippocampal neurons that were spontaneous, long lasting and high in amplitude (~50 mV). When GABA was applied exogenously, the cells exhibited the same depolarized reversal potentials (Ben-Ari et al., 1989). In order to attribute the GDPs to GABA neurotransmission, they applied GABA A receptor antagonists, bicuculline (BMI) and picrotoxin (PTX) to the cultured system. The addition of BMI and PTX caused GDPs to terminate, whereas NMDA receptor antagonists only decreased the frequency of GDP activity. Interestingly, the GABA-evoked GDPs were not present in the second week of development. Instead, spontaneous hyperpolarizing potentials were recorded in the same neurons. The hyperpolarizing potentials were also synchronous and blocked by BMI, indicating the hyperpolarization was caused by GABA. Thus, the age of the neurons seemed to be a critical determinant of the post-synaptic effect of GABA. ** **  The electrochemical gradient for chloride sets the Nernst potential (E GABA ) for the GABAergic current (Ganguly, et al., 2001). E GABA represents the difference in voltage across the membrane that would change the GABA current from excitatory to inhibitory. Ganguly et al. studied the change in the synaptic transmission of GABA using cultures of rat hippocampal neurons. They studied the effects of GABA neurotransmission on intracellular calcium concentrations over the first eighteen days of post-natal development using a calcium sensitive dye. Neurons aged 4-9 days demonstrated a rapid increase of intracellular Ca++ concentration following exogenous application of GABA. As the neurons matured, the application of GABA did not elicit increased Ca++; there was no significant response by day 13 (Ganguly et al., 2001). On day 13, KCl was added to the cell culture, which increased extracellular Ca++ levels- this indicated that the cells still had the potential to depolarize, but GABA did not mediate depolarization at this age (Fig. 1C). As the age of the cells increased, the percentage of neurons with GABA-induced increase in intracellular [Ca++] decreased non-linearly (Fig. 1B). To determine whether GABA was causing the witnessed depolarization, they applied GABA A receptor antagonists (BMI and PTX) and GABA B receptor antagonist, baclofen to the cell culture. The GABA A receptor antagonists largely blocked the increase in internal [Ca++], whereas blocking the GABA B channel had no observed effect on Ca++ concentration (Ganguly et al., 2001). The source of the intracellular Ca++ was also tested to determine whether the GABA-induced influx of calcium was through voltage-gated calcium channels (VGCCs) or whether it came from internal stores. Intracellular calcium stores were depleted using thapsigargin, which did not affect the response when GABA was exogenously applied to the cell culture. Thus, these results indicate that (1) the depolarizing effect of GABA decreases as neurons mature and (2) GABA-induced depolarization is mediated through GABA A channels, but not through GABA B channels (Ganguly et al., 2001). ** **  Chronically blocking the activity of the GABA A channel using receptor antagonists does not decrease intracellular Ca++ concentrations, whereas blocking NMDA-channels does not affect the developmental time course of excitatory to inhibitory transmission (Ganguly et al., 2001). These data indicate that (1) the hippocampal culture cells are producing and releasing GABA, (2) that GABA itself is mediating the developmental shift of E GABA ; GABAergic activity induces the transformation of GABA signaling (Ganguly et al., 2001). To test whether neuronal action potentials had any effect on the developmental switch, Ganguly et al. blocked neuronal spiking by adding tetradotoxin (TTX) to cultured neurons. As depicted in Fig. 3, TTX blockade of action potentials did not affect the time course for GABA switchover, indicating that spontaneous miniature depolarizing GABAergic synaptic currents (mGSCs) were sufficient enough to cause the switch on their own. ** **  It was previously thought that GABA depolarizing currents were due to a deficiency in available metabolic energy during development (Holmgren et al., 2010). During development, rodents have access to their mother’s milk as their only source of energy. This milk contains a large amount of fatty acids that are broken down in the liver to ketone bodies specifically DL-3-hydroxybuterate (DL-BHB). Consequently, there is an increased amount of DL-BHB, pyruvate and lactate in rat pumps. After experimentation, Holmgren and colleagues showed that high concentrations of these factors reversed the potential of GABA A receptor mediated responses at the same time as they prevented GDPs in hippocampal CA3 pyramidal neurons. Therefore, depolarizing GABA current may be associated with an increase with pyruvate and ketone bodies (Holmgren et al.,2010). ** ** Conversely, Tyzio et al. (2011), showed that the depolarizing actions of GABA are not due to increased levels of pyruvate or ketone bodies in the blood stream. The study looked at the effect of DL-BHB and pyruvate on GABA receptors in neonatal deep layers neocortical neurons and CA3 pyramidal neurons in rats. They first determined the normal physiological values of DL-BHB, pyruvate, and lactate by analyzing blood levels in newborn rats. All values were lower than those used by the Zilberter and Holmgren group. Tyzio and Colleagues also used various experimental methods including single channel recordings, patch recordings, electrophysicological recordings and 2 photon calcium imaging. ** **  In order to show that DL-BHB does not change the driving force for somatic mediated currents, recordings were made from NMDA and GABA A receptors simultaneously in both control (normal DL-BHB levels) and experimental (increased DL-BHB) groups. Tyzio et al. then conducted an experiment looking at the polarity of synaptic GABAergic responses involving local stimulation of interneurons in the stratum radiatum. Again, the results indicated that the addition of DL-BHB does not produce a reversal in polarity as determined by measuring GABA A receptor mediated post synaptic potentials (Egpsps). DL-BHB does not alter the excitatory effects of GABA. In addition, GDPs produced by GABAergic neurons under increased DL-BHB do not vary from normal GABAergic neurons both in frequency of spikes and amplitude. It is also interesting to note that normal physiological levels of pyruvate in pups did not affect GABAergic responses. However, extremely high, non-physiological levels of pyruvate were shown to cause a negative shift in Egpsps but no change in overall equilibrium potential and a decrease in the frequency of GDPs (Tyzio et al., 2011). ** **  As a result of these experiments, Tyzio and colleagues concluded that it is not the lack of energy that is causing depolarization of GABA. Rather, the depolarization may be attributed to certain chloride pumps that have a higher levels of expression neonatally. It is most likely these the KCC2/KCC1 transporters that help mediate the shift of GABA current. There exists a correlation between E GABA and increased KCC2 expression in rat hippocampal neurons (Rivera et al., 1999). Through antisense oligonucleotide inhibition of KCC2 transporters, Rivera and his team were able to show a positive shift in the reversal potential of GABA in mature pyramidal neurons. KCC2 is a co-transporter, extruding both K+ and Cl- from neurons driving up extracellular anion concentration. There are low levels of KCC2 present while the brain is still undergoing neural development and establishing its networks. During the same time there is a high incidence of NKCC1 receptors, which import chloride into the neuron (Stil et al., 2009). At this time, there is more intracellular chloride than extracellular and stimulation of the cell via GABA causes efflux of chloride anions making the cell less negative (i.e. depolarizing the cell). ** ** Since GABAergic activity drives this shift in potential, another group of experimenters attempted to examine whether an increase in KCC2 mRNA was similarly modulated. Drastic increases in KCC2 mRNA levels are found in 15 day old neurons compared to those of 3 day old neurons, as determined by RNase protection assays normalized to Beta-actin levels (Fig 2A). In addition, there is a decrease in mRNA levels by approximately 68% at day 15 if BMI and PTX were applied during neuronal development (Fig. 2B). Conversely, Ganguly and colleagues also elevated GABA exocytosis by chronically depolarizing cell cultures with KCl, showing 69+/-6% increase in mRNA KCC2 levels compared to control groups, circa day 9 (Fig. 2C). KCl addition alters the Nernst potential of chloride causing stimulations to depolarize the neuron, allowing GABA release into the synapse. KCC2 upregulation following GABAergic activity and downregulation following GABA blockade highlight GABA’s importance in shifting itself from a depolarizing to hyperpolarizing neurotransmitter. ** **  In conclusion, GABA transmission undergoes a shift from excitatory to inhibitory during early development. It has become clear to us that this shift is most likely correlated with a shift in expression of the KCC2 channel responsible for the extrusion of chloride to the extracellular environment. Additionally, there is evidence indicating there is no expression of KCC2 during GABA’s depolarizing effects (Tyzio et al as cited from Price et al., 2005; Gilbert et al., 2007; Pozas et al., 2008). Furthermore, overexpression of KCC2 in zebrafish cortical neurons was shown to have an effect on GABA polarity, GABA synapse formation and neuronal development in vivo (Reynolds et al., 2010). So is GABA really inhibitory? The answer to this question is no. GABA itself is never intrinsically inhibitory or excitatory. GABAergic activity mediates the conversion of GABA effects on the post-synaptic cell, however the post-synaptic effects of GABA are contingent upon the developmental stage of the neuron and its equilibrium potential for chloride, not the neurotransmitter molecule. **

** //Figure 1//. As presented in Ganguly et al., 2001. Fig. 1A illustrates the percent increase and time course changes in [Ca++] after exogenous application of GABA and selected channel blockers. Fig. 1B plots the percent of neurons with GABA-induced increases in intracellular [Ca++] from ages 3-19. Fig. 1C shows flourescent recordings of Ca++ in the cultured cells of young (day 7) and mature (day 13) cells. Measurements were made at (1) baseline; (2) after exogenous application of GABA; (3) after recovery of GABA-induced effects; (4) after application of KCl. **

** //Figure 2.// As presented in Ganguly et al. 2001. Fig. 2A depicts drastic increase in KCC2 mRNA levels in neuronal cultures from days 12-15 compared to levels at day 3. Fig. 2B shows a decrease in KCC2 mRNA in neurons in which GABAergic activity was chronically blocked by both PTX and BMI. Fig. 1C shows an increase in KCC2 mRNA levels when KCl induced depolarizations increased GABAergic activity of the neurons. Fig. 2D shows a summary of experiments and their effects on induction of KCC2 mRNA. **

** //Figure 3.// As presented in Ganguly et al. 2001. Addition of TTX, a blocker for action potentials, did not change the time course during which GABAergic current switched from depolarizing to hyperpolarizing. **

** References **

** Bear, M. F., Connors, B. W., & Paradiso, M. A. (2007). Neuroscience, exploring the brain. (3rd ** ** edition ed., Vol. 3). Philadelphia: Lippincott-Raven Publishers **

**Ganguly K., Schinder A. F., Wong S. T., Poo M. (2001). GABA itself promotes the developmental ** **switch of neuronal GABAergic responses from excitation to inhibition. Cell 105, 521–532. ** **doi: 10.1016/S0092-8674(01)00341-5 **


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** Karunesh, G., Schinder, F. A., Wong , T. S., & Poo, M. (2001). GABA itself promotes the ** ** developmental switch of neuronal gabaergic responses from excitation to inhibition. Cell, 105, 521-532. Retrieved from http://www.cell.com/abstract/S0092-8674(01)00341-5 **


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