The aims of the present study were 1) to explore the distribution of GABA-A channel subunits in human gliomas of various histological subtypes, and 2) to search for correlations between GABA-A channel subunits and patient survival. We showed that 17 of the 19 different GABA-A channel subunit mRNAs were present, only ρ1 and ρ3 subunits could not be detected. While no major differences in subunit mRNA levels were found between astrocytomas and oligodendrogliomas, there was a general down-regulation of GABA-A channel subunits in glioblastoma. Thus, highest mRNA levels were recorded in gliomas grade II and grade III, except for the θ subunit that showed 5–10 fold higher levels in glioblastomas.
Our findings are in agreement with previous reports demonstrating functional bindings sites for the GABA-A channel receptor in low-grade and anaplastic gliomas but not in glioblastomas 
. The lack of responsiveness to GABA in glioblastomas has been explained by either the loss of GABA-A channels or by defective channels 
. Thus, tumor cells equipped with functional GABA-A channels may be able to maintain low or intermediate proliferative activity upon physiological levels of GABA in the extracellular fluid. GABA triggered a depolarization in the majority of glioma cells and a concomitant increase of intracellular Ca2+
. Since the GABA-A channel itself is not permeable for Ca2+
ions, the Ca2+
influx has been associated with an activation of voltage-gated Ca2+
channels in the tumor cell membrane. The central role of intracellular Ca2+
as a downstream effector of GABA receptor signaling in cell proliferation has been confirmed 
The other main finding of this study is the strong co-expression of ρ2 and θ subunit proteins in gliomas grade II and the association between ρ2 subunit expression and longer patient survival in diffuse astrocytomas. In spite of its strong co-expression with the ρ2 subunit, the θ subunit did not emerge as an independent predictor of survival in the multivariate model. These findings are in agreement with the qRT-PCR results, showing an opposite trend for the θ subunit with up-regulation in glioblastomas. The prognostic impact of ρ2 subunits in diffuse astrocytomas is intriguing but is not necessarily unique for the GABA A-channel subunit families. It is possible that we failed to identify associations for other subunits in oligodendrogliomas and oligoastrocytomas, which were combined into one group in the present study due to the limited number of tumor samples. Neither can we exclude that there has been a selection bias influencing our results, since many tumor biopsies from the original clinical cohort were too small to be included in the TMA. Thus, our study provides a first link between GABA-A channel composition and survival in gliomas and supports a presumed functional role of GABA in gliomas, but the findings need to be confirmed in larger studies.
The GABA-A channel subunits exist as a family of subtypes with distinct temporal and spatial patterns of expression and distinct properties that presumably underlie a precise role for each subtype 
. The differential sensitivity to GABA is dependent on the specific subunit composition of the A-channel, and may further be influenced by intracellular proteins interacting with the channel complex and by post-translational modifications 
. Whilst the α1 subunit has frequently been the focus of studies, due to its abundance in the brain and its reliability of measurement, considerably less is known on the other subunits expressed at a lower level such as θ and the ρ2 subunits. In the brain, functional GABA-A channel receptors containing the θ subunit are formed together with α, β and γ subunits 
. Synaptic channels are activated by high GABA concentrations, whereas extrasynaptic channels can be activated by extremely low concentrations of GABA. Tumors such as gliomas are probably not exposed to high GABA concentrations and one would expect mostly tonic currents to be active in these tumors. It seems that tonic currents more or less can contain any subunit in their channel complex, whereas synaptic channels are more restricted to certain types of subunits. As such, the γ2 subunit is a major component of synaptic channels, while the α4 α5 α6 and δ subunits are part of extrasynaptic GABA A-channels and thus involved in tonic currents 
. The α1, α2 and α3 subunits can be located both at and outside of synapses and the ε and θ subunits are probably mostly extrasynaptic 
In the present study, ρ2 subunit expression was found in a relatively large number of glioma samples while no detectable mRNA levels were found by qRT-PCR for the ρ1 and ρ3 subunits. The ρ1 subunit is predominantly expressed in the retina and visual pathways, while ρ2 expression has also been found in hippocampus and amygdala 
. Rho subunits form functional homomeric GABA-A-ρ receptors (previously known as GABAC
, but can potentially also participate in heteromeric GABA-A channel formation 
. The GABA-A-ρ receptor has a number of distinctive and unique functional properties, such as a long mean opening time of the channel and slow desensitization, suggesting a more widespread function than previously thought 
. In addition, the pharmacological properties of the GABA-A-ρ receptor, including the lack of response to benzodiazepines and barbiturates, set apart this class of receptor.
We found a strong positive correlation between the expression of θ and ρ2 subunits. Expression of the θ and ρ2 subunits in brain tissue is relatively uncommon, and there is no evidence to suggest that they form a functional channel together 
. However, normal transcriptional regulation in tumor cells is disrupted and may not follow the normal “rules” for GABA-A channel subunit expression. It is therefore possible that they could have been expressed without participating in forming functional channels. Interestingly, the strongest correlation between the expression of ρ2 and θ subunits was found in gemistocytic astrocytomas. The presence of gemistocytes in a tumor has been correlated with worse prognosis 
, although the proportion of gemistocytes in the tumor does not seem to have an effect 
. The reasons for development of gemistocytes in tumors are still poorly understood, but it has been suggested that they may develop due to losing the competition for substrates with adjacent cells 
It is not known whether tumor cells are the source of GABA. Only neurons contain glutamic acid decarboxylase (GAD), the enzyme that catalyzes the decarboxylation of glutamate to GABA, and are able to produce GABA. However, astrocytes can take up GABA and there is a minor pathway not involving GAD that allows synthesis of GABA 
. Both neurons and astrocytes can release GABA, but only neurons can release high concentrations of GABA within a short time-span from synaptic vesicles at the presynaptic terminal 
The present study shows a down-regulation of subunit mRNA levels in glioblastomas, except for the θ subunit, and the presence of distinct GABA-A channel subunit proteins in gliomas grade II. The correlation between ρ2 subunits and favorable survival in diffuse astrocytomas suggests that specific subunit compositions affect clinical outcome in glioma. Taken the distinct pharmacological properties of the different GABA-A channel subtypes, this may open up for new therapeutic strategies for glioma. Our study also highlights the complexity of GABA signaling in gliomas and stresses the need for functional studies in this field.