Cloning of the GABAB Receptor Subunits B1 and B2 and their Expression in the Central Nervous System of the Adult Sea Lamprey

Abstract

In vertebrates, γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the central nervous system (CNS) acting through ionotropic (GABAA) and metabotropic (GABAB) receptors. The GABAB receptor produces a slow inhibition since it activates second messenger systems through the binding and activation of guanine nucleotide-binding proteins [G-protein-coupled receptors (GPCRs)]. Lampreys are a key reference to understand molecular evolution in vertebrates. The importance of the GABAB receptor for the modulation of the circuits controlling locomotion and other behaviors has been shown in pharmacological/physiological studies in lampreys. However, there is no data about the sequence of the GABAB subunits or their expression in the CNS of lampreys. Our aim was to identify the sea lamprey GABAB1 and GABAB2 transcripts and study their expression in the CNS of adults. We cloned two partial sequences corresponding to the GABAB1 and GABAB2 cDNAs of the sea lamprey as confirmed by sequence analysis and comparison with known GABAB sequences of other vertebrates. In phylogenetic analyses, the sea lamprey GABAB sequences clustered together with GABABs sequences of vertebrates and emerged as an outgroup to all gnathostome sequences. We observed a broad and overlapping expression of both transcripts in the entire CNS. Expression was mainly observed in neuronal somas of the periventricular regions including the identified reticulospinal cells. No expression was observed in identifiable fibers. Comparison of our results with those reported in other vertebrates indicates that a broad and overlapping expression of the GABAB subunits in the CNS is a conserved character shared by agnathans and gnathostomes.

GABA promotes survival and axonal regeneration in identifiable descending neurons after spinal cord injury in larval lampreys

“…Increased expression of the gabab1 subunit in identifiable descending neurons after SCI GABAB receptors are obligate heterodimers formed by gabab1 and gabab2 subunits (Kammerer et al, 1999). In previous work, we reported the expression of the gabab1 and gabab2 receptor subunits in identifiable descending neurons of adult sea lampreys under normal conditions (Romaus-Sanjurjo et al, 2016). Here, we used gabab1 in situ hybridization first to confirm that this receptor is also expressed in identifiable descending neurons of mature larval sea lampreys (Suppl.…”

“…Fig. 1) was conducted as previously described (Romaus-Sanjurjo et al, 2016). Briefly, brain sections were incubated with the sea lamprey gabab1 DIG-labelled probe at 70 ºC and treated with RNAse A (Invitrogen, Massachusetts, USA) in the post-hybridization washes.…”

GABA promotes survival and axonal regeneration in identifiable descending neurons after spinal cord injury in larval lampreys

“…The morpholino experiments suggest that GABA might promote regeneration by activating GABAB receptors expressed in the axotomized descending neurons. But, we cannot rule out the possibility that GABA could also promote the regeneration of descending neurons indirectly by inhibiting other cells expressing GABAB receptors, like intrinsic spinal cord neurons 39 , 42 that could have also taken the morpholino in our experiments. Our data agree with previous in vitro or developmental studies regarding the role of GABA and GABAB receptors in neurite outgrowth 67 .…”

“…GABAB receptors are obligate heterodimers formed by gabab1 and gabab2 subunits 49 . In previous work, we reported the expression of the gabab1 and gabab2 receptor subunits in identifiable descending neurons of adult sea lampreys under normal conditions 42 . Here, we used gabab1 in situ hybridization first to confirm that this receptor is also expressed in identifiable descending neurons of mature larval sea lampreys (Suppl.…”

“…In situ hybridization with a specific riboprobe for the gabab1 subunit of the sea lamprey gabab receptor (GenBank accession number KX655780; see Suppl. Figure 1 ) was conducted as previously described 42 . Briefly, brain sections were incubated with the sea lamprey gabab1 DIG-labelled probe at 70 °C and treated with RNAse A (Invitrogen, Massachusetts, USA) in the post-hybridization washes.…”