Sea lamprey brain shows evolution of neurotransmitter co-localization in vertebrates

“Dopamine and gamma-aminobutyric acid are colocalized in restricted groups of neurons in the sea lamprey brain: insights into the early evolution of neurotransmitter colocalization in vertebrates.”

Anton Barreiro-Iglesias, Verona Villar-Cervino, Ramon Anadon, and Maria Celina Rodicio

2009

In this experiment, the co-release and co-localization of neurotransmitters, such as dopamine and Ɣ-aminobutyric acid, within sea lamprey neurons were used to further understand the evolution of vertebrates.

Introduction

Sea lampreys (Petromyzon marinus), despite the complexity of their life cycle from embryo to adult, have retained many characteristics of the ancient central nervous system of vertebrates. They are members of the agnathan family of ancient vertebrates. As a result, sea lampreys are a good model organism to trace the evolution of the vertebrate nervous system (Abalo et al. 2005). Additionally, sea lampreys have also been observed to be relatively unchanged for over 500 million years (Nikitina et al. 2009). Sea lampreys are also easy to capture for studies as they have been labeled as an invasive species within the Great Lakes (Sorensen and Hoye 2007). The accessibility and origins of this model organism help it serve as a foundation for comparison when deciphering the timeline of vertebrate evolution.

Sea Lamprey (Petromyzon marinus). (Courtesy of fish.dnr.cornell.edu/nyfish/Petromyzontidae/sealamprey)

In several vertebrate models, neurotransmitters have been observed to co-release with other molecules such as peptides or nucleotides. However, the occurrence of two neurotransmitters being released is not as commonly observed. Through this particular study, it has been observed that dopamine and Ɣ-aminobutyric acid are co-released.

Dopamine is a neurotransmitter that helps to coordinate movement through the central nervous system of the organism (Abalo et al. 2005). Ɣ-aminobutyric acid is an inhibitory neurotransmitter that helps to control the excitability of the central nervous system (Meléndez-Ferro et al. 2001).

Methods

Samples of adult sea lampreys (Petromyzon marinus) were anaesthetized, and their brains were dissected out. Double immunofluorescence experiments were used with two antibodies: anti-DA and anti-GABA. To observe whether or not co-localization occurred with dopamine (DA) and Ɣ-aminobutyric acid (GABA), photomicrographs were taken of sea lamprey brain neurons. In order to determine the quantification of co-localization, the percentages of cells that were both DA (DA-ir) and GABA (GABA-ir) immunoreactive were calculated.

Results

Highest degrees of co-localization of DA and GABA were observed in the caudal rhombencephalic cerebrospinal fluid contacting population, ventral isthmus (VIS), postoptic commissure nucleus (PCN), preoptic nucleus (PON), striatum (ST) and granule-like cells of olfactory bulbs (OB) (Figure 1, red circles). At least 25% of the neurons in these labeled populations expressed co-localization of both DA and GABA. Identifying the areas in the sea lamprey brain that were both dopaminergic (dopamine activated systems) and GABAergic (GABA activated systems) was helpful in elucidating the general scheme of organization within the organism’s neurons.

Figure 1. Schematic drawing of a lateral view of the brain of the sea lamprey showing the distribution of double-labelled DA-ir/GABA-ir cells (black dots) in comparison with the distribution of DA-ir cells (grey dots). This is the general organization of the GABAergic and dopaminergic systems in the adult sea lamprey.

Photomicrographs show the co-localization observed in the lamprey neurons (Figures 2 and 3).

Figure 2. Photomicrographs of transverse sections of the brain showing DA (red) and GABA (green) immunoreactivities. (A) Immunoreactivities of both DA and GABA in the caudal rhombencephalon appear yellow (A’) Immunoreactivity of DA alone in the caudal rhombencephalon (A’’) Immunoreactivity of GABA alone in the caudal rhombencephalon (B-D) Closeup of the arrowhead in A-A’’ which are focused on double labeled thin fibres (E) Immunoreactivities of both DA and GABA in the ventral isthmus appear yellow (E’) Immunoreactivity of DA alone in the ventral isthmus (E’’) Immunoreactivity of GABA alone in the ventral isthmus (F) Immunoreactivities of both DA and GABA in the ventral hypothalamus appear yellow (G) Immunoreactivity of DA alone in the ventral hypothalamus, close-up (E’’) Immunoreactivity of GABA alone in the ventral hypothalamus, close-up

Photomicrographs of transverse sections of the rostal diencephalon and telencephalon showing DA (red) and GABA (green) immunoreactivities. (A) Immunoreactivities of both DA and GABA in the rostal diencephalon appear yellow (A’) Immunoreactivity of DA alone in the rostal diencephalon (A’’) Immunoreactivity of GABA alone in the rostal diencephalon (B) Immunoreactivities of both DA and GABA in the striatum appear yellow; cells are cerebrospinal fluid contacting (B’) Immunoreactivity of DA alone in the striatum; cells are cerebrospinal fluid contacting (B’’) Immunoreactivity of GABA alone in the striatum; cells are cerebrospinal fluid contacting (C) Immunoreactivities of both DA and GABA in the striatum appear yellow; cells are not cerebrospinal fluid contacting (C’) Immunoreactivity of DA alone in the striatum; cells are not cerebrospinal fluid contacting (C’’) Immunoreactivity of GABA alone in the striatum; cells are not cerebrospinal fluid contacting (D)(E)(E’)(E’’)

Figure 3. Photomicrographs of transverse sections of the rostal diencephalon and telencephalon showing DA (red) and GABA (green) immunoreactivities. (A) Immunoreactivities of both DA and GABA in the rostal diencephalon appear yellow (A’) Immunoreactivity of DA alone in the rostal diencephalon (A’’) Immunoreactivity of GABA alone in the rostal diencephalon (B) Immunoreactivities of both DA and GABA in the striatum appear yellow; cells are cerebrospinal fluid contacting (B’) Immunoreactivity of DA alone in the striatum; cells are cerebrospinal fluid contacting (B’’) Immunoreactivity of GABA alone in the striatum; cells are cerebrospinal fluid contacting (C) Immunoreactivities of both DA and GABA in the striatum appear yellow; cells are not cerebrospinal fluid contacting (C’) Immunoreactivity of DA alone in the striatum; cells are not cerebrospinal fluid contacting (C’’) Immunoreactivity of GABA alone in the striatum; cells are not cerebrospinal fluid contacting (D) Overall preoptic nucleus (E) Immunoreactivities of both DA and GABA in the preoptic nucleus appear yellow (E’) Immunoreactivity of DA alone in the preoptic nucleus (E’’) Immunoreactivity of GABA alone in the preoptic nucleus

Photomicrographs also showed the co-localization of neurotransmitters in terminals and fibres (Figures 4 and 5). Terminals that were observed to have both DA and GABA localization were identified as double-labelled. Overall, double-labelled terminals and fibres represented a small proportion of the co-localized neurons observed in this study.

Figure 4. Photomicrographs of transverse sections of the rostal diencephalon and telencephalon showing DA (red) and GABA (green) immunoreactivities. (A) Immunoreactivities of both DA and GABA in the olfactory bulb appear yellow (A’) Immunoreactivity of DA alone in the olfactory bulb (A’’) Immunoreactivity of GABA alone in the olfactory bulb

Figure 5. Photomicrographs of transverse sections of the brain showing DA (red) and GABA (green) immunoreactivities in fibres or terminals (arrowheads).

Discussion

In looking at the results of this study on sea lamprey neurons in comparison to neurotransmitter localization in mammals and other vertebrates, it is possible to somewhat trace the timeline of vertebrate evolution. For example, co-localization of DA and GABA is observed in the striatum of both mammals and the sea lamprey brain. While the sources of these neurotransmitters are observed to be different, the path of innervation of these neurotransmitters is very similar, as seen by depletion experiments. Thus, these findings show that there is a conservation of “neurochemical organization” between sea lampreys and mammals. Additionally, with evidence that DA and GABA are both found in the terminals and fibres of different regions of the sea lamprey brain, there is further support for the hypothesis that co-release of these two neurotransmitters occurs.

Previous studies have shown that DA and GABA are co-localized in the neurons of certain invertebrates and vertebrates. In fact, a previous study already proved the co-localization of both DA and GABA in some spinal cord neurons of sea lampreys (Barreiro-Iglesias et al. 2008). Essentially, this study was an extension of previous work that was able to further delve into the organization of the sea lamprey’s brain.

Based on the results of this study that a relatively significant percentage of the nuclei of sea lamprey brains express the co-localization of both DA and GABA, it can be concluded that this observed co-localization occurred early in the evolution of vertebrates. It is important to note the long-standing and relatively unchanging existence of the sea lamprey as part of the agnathan family of vertebrates. Additionally, this co-localization is evidence that the sea lamprey neural system is more complex than originally thought. Further studies have also shown that co-localization can occur with other neurotransmitters such as serotonin and glycine, further adding to the complexity and expanse of this neuronal system among early and modern vertebrates (Villar-Cerviño et al. 2009).

Conclusions and Implications

Through the better understanding of the mechanism and organization of co-localization obtained through this study, the evolution of the vertebrate neuronal system becomes clearer. Knowing that co-localization is not limited to just the dopamine and GABA neurotransmitters, it is possible to conclude that the sea lamprey brain and the vertebrate neuronal system, by default, is more complex than previously thought. However, the data and insight obtained from this study further elucidates the purpose and function of co-localized neurotransmitter release. This is especially useful in understanding the neurochemical function in motor and brain control of the sea lamprey. By knowing more about the co-release of neurotransmitters, it is possible to gain a stronger understanding of how this affects receptor function, plasticity among neurons, and motor function.

Further directions of this study could include looking at the exact function of each neurotransmitter in the co-localized release of DA and GABA. Looking at receptor function and the effects of other neurotransmitters’ release could be other directions this study could pursue. Additionally, it would be beneficial for this particular study to examine whether or not there are differences in the co-release of neurotransmitters between larval and adult organisms of this species. Differences between species of sea lampreys is also something to take into consideration.

References

Abalo, Xesús M. et al. “Development of the dopamine-immunoreactive system in the     central nervous system of the sea lamprey.” Brain Research Bulletin. 66 (2005): 560 – 564. Print.

Barreiro-Iglesias, Antón et al. “Development and organization of the descending serotonergic brainstem-spinal projections in the sea lamprey.” Journal of Chemical Neuroanatomy. 36 (2008): 77 – 84. Print.

Meléndez-Ferro, Miguel et al. “GABA immunoreactivity in the olfactory bulbs of the adult sea lamprey Petromyzon marinus L.” Brain Research. 893 (2001): 253 – 260. Print.

Nikitina, Natalya et al. “The Sea Lamprey Petromyzon marinus: A Model for Evolutionary and Developmental Biology.” Emerging Model Organisms. Print.

Sorensen, P.W. and T.R. Hoye. “A critical review of the discovery and application of migratory pheromone in an invasive fish, the sea lamprey Petromyzon marinus L.” Journal of Fish Biology. 71 (2007): 100 – 114. Print.

Villar-Cerviño, Verona et al. “Development of glycine immunoreactivity in the brain of the sea lamprey: Comparison with ɣ- aminobutyric acid immunoreactivity.” The Journal of Comparative Neurology. 512 (2009): 747 – 767. Print.