Lamprey Brain Research Articles

Development of the Lamprey Central Nervous System, with Reference to Vertebrate Evolution

Vertebrate brains are highly organized structures that show remarkable diversity throughout animal groups. The agnathans, which diverged from the gnathostomes early in the evolution of the vertebrates, occupy a key phylogenetic position from which to clarify the origin and evolution of the brain. We studied the developing lamprey brain and compared its developmental plan with that of the gnathostomes, in order to reconstruct the evolutionary processes of the vertebrate brain. We found that the lamprey brain has the basic molecular mechanisms necessary to form neuromeric compartments, and that its mesencephalon and diencephalon exhibit conserved morphological and molecular features. Conversely, the telencephalon and metencephalon display lamprey-specific developmental mechanisms. Thus, the molecular program of the nervous system is thought to have improved in the gnathostome lineage. Changes in the expression domains of some regulatory genes might have facilitated the evolution of the vertebrate central nervous system.

Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys

There is controversy about whether axotomized neurons undergo death or only severe atrophy after spinal cord injury (SCI) in mammals. Lampreys recover from complete spinal transection, but only about half of the severed spinal-projecting axons regenerate through the site of injury. The fates of the unregenerated neurons remain unknown, and until now death of axotomized spinal-projecting neurons has not been described in the lamprey brain. We now report that in animals allowed to survive for 12 or more weeks after spinal cord transection, several identified reticulospinal (RS) neurons were missing in Nissl-stained or neurofilament-immunostained brain whole mounts. At earlier times, these neurons were swollen and pale in Nissl-stained preparations. Retrograde fluorescent labeling from the site of transection combined with TUNEL histochemistry suggested that neuronal death, including that of the identified RS neurons, began in animals 4 weeks posttransection, reaching a peak at 12-16 weeks. This was not seen in untransected animals. The TUNEL positivity suggests that some cells were dying by apoptosis. Of special interest, among the identified neurons, this delayed cell death was restricted to neurons that at earlier posttransection times have a low probability of regeneration. These data show that SCI induces delayed cell death in lamprey spinal-projecting neurons and suggest that the reason why some neurons are "bad regenerators" is that they are already undergoing apoptotic cell death. Thus protection from apoptosis may be necessary in order to enhance axonal regeneration after SCI.

Distribution of glycine immunoreactivity in the brain of adult sea lamprey (Petromyzon marinus). Comparison with γ‐aminobutyric acid

Distribution of glycine immunoreactivity in the brain of adult sea lamprey (Petromyzon marinus). Comparison with γ‐aminobutyric acid

Neuroendocrine and behavioral responses to weak electric fields in adult sea lampreys ( Petromyzon marinus)

We characterized the behavioral and neuroendocrine responses of adult sea lampreys ( Petromyzon marinus) to weak electric fields. Adult sea lampreys, captured during upstream spawning migration, exhibited limited active behaviors during exposure to weak electric fields and spent the most time attached to the wall of the testing arena near the cathode (−). For adult male sea lampreys, exposure to weak electric fields resulted in increased lamprey (l) GnRH-I mRNA expression but decreased lGnRH-I immunoreactivities in the forebrain, and decreased Jun (a neuronal activation marker) mRNA levels in the brain stem. Similar effects were not observed in the brains of female sea lampreys after weak electric field stimulation. The influence of electroreception on forebrain lGnRH suggests that electroreception may modulate the reproductive systems in adult male sea lampreys. The changes in Jun expression may be associated with swimming inhibition during weak electric field stimulation. The results for adult sea lampreys are the opposite of those obtained using parasitic-stage sea lampreys, which displayed increased activity during and after cathodal stimulation. Our results demonstrate that adult sea lampreys are sensitive to weak electric fields, which may play a role in reproduction. They also suggest that electrical stimuli mediate different behaviors in feeding-stage and spawning-stage sea lampreys.

Distribution of glycine immunoreactivity in the brain of adult sea lamprey (Petromyzon marinus). Comparison with γ‐aminobutyric acid

The distribution of glycinergic cells in the brain of nonmammalian vertebrates is still unknown. Lampreys are the most primitive extant vertebrates, and they may provide important data on the phylogeny of this system. Here, we studied for the first time the distribution of glycine immunoreactivity in the sea lamprey brain and compared it with gamma-aminobutyric acid (GABA)-ergic populations. Most glycine-immunoreactive neurons were found at midbrain and hindbrain levels, and most of these cells did not exhibit GABA immunoreactivity. We describe glycine-immunoreactive cell populations in the olfactory bulbs, the preoptic nucleus, and the thalamus of the sea lamprey, which is in striking contrast to their lack in the mammalian forebrain. We also observed glycine-immunoreactive populations in the optic tectum, the torus semicircularis and the midbrain tegmentum, the isthmus, the octavolateral area, the dorsal column nucleus, the abducens nucleus, the trigeminal motor nucleus, the facial motor nucleus, and the rhombencephalic reticular formation. In these populations, colocalization with GABA was observed in only some cells of the tegmental M5 nucleus, ventral isthmus, medial octavolateral nucleus, dorsal column nucleus, and lateral reticular region. The present results allow us to conclude that the distribution of glycine-immunoreactive cells changed notably from lamprey to mammals, with a decrease in glycinergic populations in the forebrain and a specialization of brainstem cell groups. Although knowledge of the glycinergic populations in lampreys is important for understanding the early evolution of this system, there is a notable gap of information regarding its organization in brains of other nonmammalian vertebrates.

Distribution of adrenomedullin-like immunoreactivity in the brain of the adult sea lamprey

Adrenomedullin (AM) is a neuropeptide widely distributed in vertebrates. In jawed vertebrates it has been localized in distinct regions of the central nervous system by means of antisera against human AM because the molecule seems to be well conserved across species. In this study, we have analyzed the localization of AM-like immunoreactive (AM-ir) cell bodies and fibers throughout the brain of the adult sea lamprey Petromyzon marinus, by using immunohistochemistry. Several AM-ir cell populations were found in the basal plate of the secondary prosencephalon, being more numerous in the hypothalamus, as well as two in the diencephalon and one in the mesencephalon; in addition two cell populations were found in the rhombencephalic alar plate, one in the isthmic region and other in the nucleus of the solitary tract. Immunolabeled fibers were widespread throughout the lamprey brain, but were more abundant in the basal plate. Of particular interest was the conspicuous innervation of the striatum by AM-ir fibers. In addition, our results indicate that AM-ir cells and fibers are present in the lamprey hypothalamo-neurohypophyseal system, suggesting that AM plays some important role in the control of pituitary gland function.

Reticulospinal neurons in anamniotic vertebrates: A celebration of Alberto Stefanelli's contributions to comparative neuroscience

Over the past 76 years Alberto Stefanelli has successfully used a comparative approach to study the nervous system. His main research focus during that time has been on identifiable reticulospinal neurons including Müller and Mauthner neurons found in anamniotic vertebrates. Born in Venice, Italy in 1908, Professor Stefanelli pursued most of his academic career at the University of Rome, where he retired as Chair of Comparative Anatomy in 1978.His seminal work on the constancy in number and position of giant identifiable reticulospinal neurons in the brains of larval and adult lampreys, and his assertion that only a subset of these neurons were Müller cells, provided the framework in which subsequent authors have refined our understanding of the cellular anatomy, axonal projections, physiology, and function of Müller cells in the control of movement. Stefanelli has also provided the most comprehensive study to date of the Mauthner cell and its axon cap. His description of the differences in axon cap structure among many fishes and amphibians and his use of the “morpho-ecological” approach to determine Mauthner cell function has provided the basis for future studies on the neuronal basis of behavior and its evolution.As Professor Stefanelli approaches his 100th birthday, we celebrate his scientific contributions to comparative neuroscience with a biographical sketch of his life, an overview of his scientific accomplishments, and our view of how his comparative studies continue to contribute to our understanding of the nervous system.

A lamprey striatal brain slice preparation for patch-clamp recordings

Striatum, the input layer of the basal ganglia is important for functions such as the selection of motor behaviour. The lamprey, a lower vertebrate, is particularly well suited as a model system for the control of motor functions as its central nervous system is similar to that of higher vertebrates and exhibits a lower level of complexity. Therefore, studies in lamprey preparations enable cellular and synaptic mechanisms to be correlated with behaviour. The lamprey brain slice preparation presented has been developed to study the striatal microcircuits and input/output systems with patch-clamp recordings. The method involves dissection of the central nervous system, brain slice preparation, identification of the striatum, visual identification of striatal neurons and patch-clamp recordings. By combining studies in the slice preparation presented here and other lamprey preparations such as the semi-intact lamprey, we will be able to correlate striatal mechanisms on the cellular, synaptic and network levels with striatal output and motor behaviour. The method can be adapted to produce similar slice preparations from other areas of the lamprey brain.

RFamide Peptides Inhibit the Expression of Melanotropin and Growth Hormone Genes in the Pituitary of an Agnathan, the Sea Lamprey, Petromyzon marinus

Neuropeptides with the Arg-Phe-amide motif at their C termini (RFamide peptides) were identified in the brains of several vertebrates, and shown to have important physiological roles in neuroendocrine, behavioral, sensory, and autonomic functions. The present study identified RFamide peptides, which are teleost prolactin-releasing peptide (PrRP) homologs, in the sea lamprey, Petromyzon marinus and characterized their effect on the release of pituitary hormones in vitro. Two RFamide peptides (RFa-A and RFa-B) were isolated from an acid extract of sea lamprey brain, including hypothalamus by Sep-Pak C18 cartridge, affinity chromatography using anti-salmon PrRP serum, and reverse-phase HPLC on an ODS-120T column. Amino acid (aa) sequences and mass spectrometric analyses revealed that RFa-A and RFa-B consist of 25 and 20 aa, respectively, and have 75% sequence identity within the C-terminal 20 aa. The RFa-B cDNA encoding a preprohormone of 142 aa was cloned from the lamprey brain, and the deduced aa sequence from positions 48-67 was identical to the sequence of RFa-B. However, the preprohormone does not include an aa sequence similar to the RFa-A sequence. Cell bodies, which were immunoreactive to anti-salmon PrRP serum, were located in the periventricular arcuate nucleus, ventral part of the hypothalamus, and immunoreactive fibers were abundant from the hypothalamus to the brain. A small number of immunoreactive fibers were detected in the dorsal half of the rostral pars distalis of the pituitary, close to the GH-producing cells. In addition, anti-salmon PrRP immunoreactivities were observed in the pars intermedia, corresponding to melanotropin cells. Likewise, signal of RFa-B mRNA was detected not only in the brain but also in the pars intermedia. The synthetic RFa-A and -B inhibited GH mRNA expression in a dose-dependent fashion in vitro, which is comparable to the inhibitory effect of teleost PrRP on GH release. Both RFa-A and -B also inhibited the expression of proopiomelanotropin mRNA, but no effects were observed in the expression of proopiocortin and gonadotropin beta mRNAs. The results indicate that RFamide peptides, which are teleost PrRP homologs, are present in the hypothalamus and pituitary of sea lamprey, and may be physiologically involved in the inhibition of GH and melanotropin release in the sea lamprey pituitary.

Evolutionary origin and divergence of PQRFamide peptides and LPXRFamide peptides in the RFamide peptide family

Among the RFamide peptide groups, PQRFamide peptides, such as neuropeptide FF (NPFF) and neuropeptide AF (NPAF), share a common C-terminal Pro-Gln-Arg-Phe-NH(2) motif. LPXRFamide (X = L or Q) peptides, such as gonadotropin-inhibitory hormone (GnIH), frog growth hormone-releasing peptide (fGRP), goldfish LPXRFamide peptide and mammalian RFamide-related peptides (RFRPs), also share a C-terminal Leu-Pro-Leu/Gln-Arg-Phe-NH(2) motif. Such a similar C-terminal structure suggests that these two groups may have diverged from a common ancestral gene. In this study, we sought to clarify the evolutionary origin and divergence of these two groups, by identifying novel RFamide peptides from the brain of sea lamprey, one of only two extant groups of the oldest lineage of vertebrates, Agnatha. A novel lamprey RFamide peptide was identified by immunoaffinity purification using the antiserum against LPXRFamide peptide. The lamprey RFamide peptide did not contain a C-terminal LPXRFamide motif, but had the sequence SWGAPAEKFWMRAMPQRFamide (lamprey PQRFa). A cDNA of the precursor encoded one lamprey PQRFa and two related peptides. These related peptides, which also had the C-terminal PQRFamide motif, were further identified as mature endogenous ligands. Phylogenetic analysis revealed that lamprey PQRFamide peptide precursor belongs to the PQRFamide peptide group. In situ hybridization demonstrated that lamprey PQRFamide peptide mRNA is expressed in the regions predicted to be involved in neuroendocrine and behavioral functions. This is the first demonstration of the presence of RFamide peptides in the agnathan brain. Lamprey PQRFamide peptides are considered to have retained the most ancestral features of PQRFamide peptides.

Dynamic expression of the LIM-homeodomain gene Lhx15 through larval brain development of the sea lamprey ( Petromyzon marinus)

LIM-homeodomain genes encode a family of transcription factors with highly conserved roles in the patterning and regionalisation of the vertebrate brain. The expression of one of those genes, Lhx15, in the embryonic lamprey brain, characterises precise functional subdivisions. In order to analyse the non-embryonic development of the lamprey brain, we chose this gene to perform in situ hybridisations in Petromyzon marinus larvae of different ages. We demonstrate the usefulness of Lhx15 to follow the development and morphogenesis of brain structures and show the dynamical expression of this gene through time. Furthermore, we provide evidence for the evolutionary conservation of the expression of this gene in the spinal cord, notochord and urogenital system.

Distribution of neuropeptide FF-like immunoreactive structures in the lamprey central nervous system and its relation to catecholaminergic neuronal structures

The neuropeptide FF (NPFF) is an octapeptide of the RFamide-related peptides (FaRPs) that was primarily isolated from the bovine brain. Its distribution in the CNS has been reported in several mammalian species, as well as in some amphibians. Therefore, in order to gain insight in the evolution on the expression pattern of this neuropeptide in vertebrates, we carried out an immunohistochemical study in the sea lamprey, Petromyzon marinus. The distribution of NPFF-like-immunoreactive (NPFF-ir) structures in the lamprey brain is, in general, comparable to that previously described in other vertebrate species. In lamprey, most of the NPFF-ir cells were found in the hypothalamus, particularly in two large populations, the bed nucleus of the tract of the postoptic commissure and the tuberomammillary area. Numerous NPFF-ir cells were also observed in the rostral rhombencephalon, including a population in the dorsal isthmic gray and the reticular formation. Additional labeled neurons were found inside the preoptic region, the parapineal vesicle, the periventricular mesencephalic tegmentum, the descending trigeminal tract, the nucleus of the solitary tract, as well as in the gray matter of the spinal cord. The NPFF-ir fibers were widely distributed in the brain and the spinal cord, being, in general, more concentrated throughout the basal plate. The presence of NPFF-ir fibers in the lamprey neurohypophysis suggests that the involvement of NPFF-like substances in the hypothalamo-hypophyseal system had emerged early during evolution.

Afferents of the lamprey optic tectum with special reference to the GABA input: Combined tracing and immunohistochemical study

The optic tectum in the lamprey midbrain, homologue of the superior colliculus in mammals, is important for eye movement control and orienting responses. There is, however, only limited information regarding the afferent input to the optic tectum except for that from the eyes. The objective of this study was to define specifically the gamma-aminobutyric acid (GABA)-ergic projections to the optic tectum in the river lamprey (Lampetra fluviatilis) and also to describe the tectal afferent input in general. The origin of afferents to the optic tectum was studied by using the neuronal tracer neurobiotin. Injection of neurobiotin into the optic tectum resulted in retrograde labelling of cell groups in all major subdivisions of the brain. The main areas shown to project to the optic tectum were the following: the caudoventral part of the medial pallium, the area of the ventral thalamus and dorsal thalamus, the nucleus of the posterior commissure, the torus semicircularis, the mesencephalic M5 nucleus of Schober, the mesencephalic reticular area, the ishtmic area, and the octavolateral nuclei. GABAergic projections to the optic tectum were identified by combining neurobiotin tracing and GABA immunohistochemistry. On the basis of these double-labelling experiments, it was shown that the optic tectum receives a GABAergic input from the caudoventral part of the medial pallium, the dorsal and ventral thalamus, the nucleus of M5, and the torus semicircularis. The afferent input to the optic tectum in the lamprey brain is similar to that described for other vertebrate species, which is of particular interest considering its position in phylogeny.

In situ Characterization of Gonadotropin- Releasing Hormone-I, -III, and Glutamic Acid Decarboxylase Expression in the Brain of the Sea Lamprey, Petromyzon marinus

The distribution of lamprey gonadotropin-releasing hormone (GnRH)-I and -III has been extensively characterized by immunocytochemistry in the forebrain of the sea lamprey, Petromyzon marinus. However, the cellular location of lamprey GnRH-III mRNA expression by in situ hybridization in the lamprey brain has not been determined. We show for the first time the location of expression of lamprey GnRH-III, as well as provide a more comprehensive in situ study of lamprey GnRH-I and glutamic acid decarboxylase (GAD; GABA-synthesizing enzyme) mRNA expression in the brain of the lamprey in different reproductive life stages. Colorimetric and dual-label fluorescent amplification methods of in situ hybridization were used on brain tissue sections of adult, juvenile, and larval sea lamprey. In each life stage of the lamprey, expression of lamprey GnRH-I was shown in the preoptic area (POA) and the hypothalamus forming the characteristic arc-like cell population extending from the preoptic nucleus (NPO) to the neurohypophysis. Lamprey GnRH-III expression was also seen in the POA of each life stage in close proximity to lamprey GnRH-I mRNA containing neurons. GAD expression was shown in distinct cell clusters in and around the POA, in the olfactory bulb, in the dorsal thalamus beneath the habenular region, and also in the ventral-medial hypothalamus stretching from the periventricular region to the anterior portion of the rhombencephalon. Using dual-label in situ hybridization, we have shown that lamprey GnRH-I and -III mRNA are colocalized in the same cells in the POA in adult lampreys. Dual-label in situ hybridization also showed close proximity of GAD mRNA containing neurons and GnRH containing neurons in the POA. These data suggest that γ-aminobutyric acid (GABA) may directly affect GnRH release in the brain of the sea lamprey.

In vitro and in vivo effects of GABA, muscimol, and bicuculline on lamprey GnRH concentration in the brain of the sea lamprey ( Petromyzon marinus)

γ-Aminobutyric acid (GABA) is a neurotransmitter with a demonstrated neuroregulatory role in reproduction in most representative species of vertebrate classes via the hypothalamus. The role of GABA on the hypothalamus–pituitary axis in lampreys has not been fully elucidated. Recent immunocytochemical and in situ hybridization studies suggest that there may be a neuroregulatory role of GABA on the gonadotropin-releasing hormone (GnRH) system in lampreys. To assess possible GABA–GnRH interactions, the effects of GABA and its analogs on lamprey GnRH in vitro and in vivo were studied in adult female sea lampreys ( Petromyzon marinus). In vitro perfusion of GABA and its analogs at increasing concentrations (0.1–100 μM) was performed over a 3-h time course. There was a substantial increase of GnRH-I and GnRH-III following treatment of muscimol at 100 μM. In in vivo studies, GABA or muscimol injected at 200 μg/kg significantly increased lamprey GnRH concentration in the brain 0.5 h after treatment compared to controls in female sea lampreys. No significant change in lamprey GnRH-I or GnRH-III was observed following treatment with bicuculline. These data provide novel physiological data supporting the hypothesis that GABA may influence GnRH in the brain of sea lamprey.

Reelin immunoreactivity in the adult sea lamprey brain

The expression of reelin, a large extracellular matrix glycoprotein, was studied in the brain of pre-spawning adult sea lampreys by immunohistochemistry using two monoclonal antibodies against this protein. Reelin immunoreactive (reln-ir) neurons were observed in the olfactory bulb, and pallial and subpallial regions in the telencephalon. In the diencephalon, reln-ir cells were observed in some hypothalamic nuclei, in the nucleus of Bellonci, and in the habenula. In the mesencephalon, this protein was detected in several nuclei related with the centrifugal visual system, although the optic tectum was devoid of immunoreactivity. The hindbrain showed several nuclei with immunopositive neurons, including the branchiomeric nerve motor nuclei and also some groups of non-giant cells of the reticular formation. The rostral spinal cord showed some immunopositive neurons mainly located in lateral and ventral positions. Overall, the pattern of distribution of reelin in the adult sea lamprey correlates with the previously reported in other adult vertebrates. Furthermore, the wide distribution of reelin in the adult lamprey brain is consistent with a possible existence of different roles for this protein not related with development in the central nervous system (CNS) of vertebrates (i.e. neuronal plasticity and/or maintenance).

Reelin immunoreactivity in the larval sea lamprey brain

In order to analyze the presence of a reelin-like protein in the brain of a primitive vertebrate with a laminar-type brain, such as the sea lamprey, Western blot and immunohistochemical approaches were employed by using the G10 and 142 reelin-specific monoclonal antibodies. Western blots of lamprey brain extracts showed bands of about 400 kDa, 180 kDa and others below 100 kDa; similar bands were observed in samples from rat cerebellum. In different larval stages there was a prominent reelin immunolabeling associated with the olfactory bulb, pallial regions, habenula, hypothalamus and optic tectum. In addition, the olfactory and optic tracts, as well as the afferent and efferent (fasciculus retroflexus) tracts of the habenular ganglion, also showed immunopositivity in these stages. Interestingly, the highest level of labeling was observed in premetamorphic larvae, just prior to entering the metamorphic stage. These data indicate that reelin expression is also prominent in brains of primitive vertebrates without layered cortical regions, suggesting that some physiological roles of reelin not related to the regulation of neuronal migration in layered cortical regions (i.e. involvement in axon pathfinding, synaptogenesis, dendritic arborization and neuronal plasticity) might have appeared earlier in evolution.

Reports and Surveys

I. ADVANCED CYBORG (BIOLOGICAL ROBOT)Whilst research continues to produce robots that mimic nature's creatures, scientists at the Northwestern University in Chicago, USA have developed a robot that is controlled by the brain of a sea lamprey. This is therefore an example of a biological robot, perhaps the first of many that will be developed.

Cognitive science fiction

Cognitive science looks more like science fiction in a US lab where scientists are working on a new generation of cyborg. Half-fish, half-robot, their creation is capable of responding to light. The researchers connect the brainstem of a lamprey to a small robot on wheels, tapping into the neural network normally responsible for the lamprey's orienting reflex. When the robot detects light, it sends an electrical signal to the disembodied brain, and the brain responds by sending a signal to the wheels, causing cyborg to move towards or away from the light source. As Sandro Musso-Ivaldi, at Northwestern University, explained to TICS, ‘the robot generates stimuli for the vestibular nerves of the lamprey and receives motor commands from the reticular neurons. The final outcome is a sensory–motor behavior of the robot, organized by the connections in the lamprey's brain’. The team hope to use their neuro-robotic system to find out about the computational properties of synaptic plasticity. Such advances in cybernetics should also speed the development of brain-controlled prostheses. H.J.B

The Distribution of Melanin-Concentrating Hormone in the Lamprey Brain

In addition to its novel, colour-regulating hormonal role in teleosts, the melanin-concentrating hormone (MCH) serves as a neuromodulatory peptide in all vertebrate brains. In gnathostome vertebrates, it is produced in several neuronal cell groups in the hypothalamus. The present work examines the organisation of the MCH system in the brain of lampreys, which separated from gnathostome vertebrates at an early stage in evolution. In all three lamprey genera examined—Petromyzon, Lampetra, and Geotria spp.—MCH perikarya were found in one major anatomical site, the periventricular dorsal hypothalamic nucleus of the posterior hypothalamus. Axons from these cell bodies projected medially into the ventricular cavity, and laterally to the neuropile of the lateral hypothalamus. From here, they extended anteriorly and posteriorly to the fore- and hindbrain. Other fibres extended dorsomedially to the habenular nucleus. In Lampetra, but not in Petromyzon, MCH fibres were seen in the pituitary neurohypophysis, most prominantly above the proximal pars distalis. The hypothalamic region in which the MCH perikarya are found forms part of the paraventricular organ (PVO), which is rich in monoamines and other neuropeptides. The association of MCH neurones with the PVO, which occurs also in many other nonmammalian vertebrates, may reflect the primary location of the MCH system. These MCH neurones were present in ammocoetes, postmetamorphic juveniles, and adults. They were more heavily granulated in adults than in young lampreys but showed no marked change in secretory appearance associated with metamorphosis or experimental osmotic challenge to indicate a role in feeding or osmoregulation. In sexually maturing Lampetra fluviatilis, however, a second group of small MCH neurones became detectable in the telencephalon, suggesting a potential role in reproduction and/or behaviour.