Manuel A. Pombal

Prosomeric map of the lamprey forebrain based on calretinin immunocytochemistry, nissl stain, and ancillary markers

The structural organization of the lamprey extratelencephalic forebrain is re‐examined from the perspective of the prosomeric segmental paradigm. The question asked was whether the prosomeric forebrain model used for gnathostomes is of material advantage for interpreting subdivisions in the lamprey forebrain. To this aim, the main longitudinal and transverse landmarks recognized by the prosomeric model in other vertebrates were identified in Nissl‐stained lamprey material. Lines of cytoarchitectural discontinuity and contours of migrated neuronal groups were mapped in a two‐dimensional sagittal representation and were also classified according to their radial position. Immunocytochemical mapping of calretinin expression in adjacent sections served to define particular structural units better, in particular, the dorsal thalamus. These data were complemented by numerous other chemoarchitectonic observations obtained with ancillary markers, which identified additional specific formations, subdivisions, or boundaries. Emphasis was placed on studying whether such chemically defined neuronal groups showed boundaries aligned with the postulated inter‐ or intraprosomeric boundaries. The course of diverse axonal tracts was studied also with regard to their prosomeric topography. This analysis showed that the full prosomeric model applies straightforwardly to the lamprey forebrain. This finding implies that a common segmental and longitudinal organization of the neural tube may be primitive for all vertebrates. Interesting novel aspects appear in the interpretation of the lamprey pretectum, the dorsal and ventral thalami, and the hypothalamus. The topologic continuity of the prosomeric forebrain regions with evaginated or non‐evaginated portions of the telencephalon was also examined. J. Comp. Neurol. 414:391–422, 1999.

Distribution of choline acetyltransferase-immunoreactive structures in the lamprey brain

The distribution of cholinergic neurons and fibers was studied immunohistochemically in the brain of two species of lampreys (Petromyzon marinus and Lampetra fluviatilis), by using an antiserum against choline acetyltransferase (ChAT). The results obtained in both species were similar, but there appeared some interspecies differences. In the forebrain, cholinergic cells were present in the striatum, preoptic region, paraventricular nucleus, pineal and parapineal organs, habenula, and pretectum. The cranial nerve motoneurons (III, IV, V, VI, VII, IX, and X), the first and second spino‐occipital nerves (so), and the ventral horn of the spinal cord showed a strong ChAT immunoreactivity. Additional cholinergic neurons were observed: the mesencephalic M5 nucleus of Schober, two different cell populations in the isthmic region, the efferent component of the eighth nerve, putative preganglionic parasympathetic cells, cells in the solitary tract nucleus, and the rhombencephalic reticular formation. Cholinergic fibers were widely distributed in the brain. Comparison with previous studies in other vertebrates suggests that major cholinergic pathways, like tectal innervation from the isthmic region, are also present in lampreys. Of particular interest was the prominent projection to the neurohypophysis from cholinergic neurons in the preoptic region and paraventricular nucleus. Present data were analyzed within the segmental paradigm, as was previously done in other vertebrates. Our results reveal that the organization of many cholinergic systems in the lamprey as, for example, in the striatal, preoptic, and isthmic regions, comprises features of the anamniote brain that remain common to all living amniotes studied so far, thus being conservative to a surprisingly high degree. Therefore, the distribution of ChAT‐immunoreactive structures in the lamprey brain is, in general, comparable to that previously described in other vertebrate species. J. Comp. Neurol. 431:105–126, 2001.

Afferents of the lamprey striatum with special reference to the dopaminergic system: a combined tracing and immunohistochemical study.

The origin of afferents to the striatum in lamprey (Lampetra fluviatilis) was studied by using fluorescein‐coupled dextran‐amines (FDA). Injection of FDA into the striatum retrogradely labeled several cell populations in the forebrain and the rostral rhombencephalon. No retrograde labeled cells were seen in the mesencephalon.

Diencephalic projection to reticulospinal neurons involved in the initiation of locomotion in adult lampreys Lampetra fluviatilis

Morphological and electrophysiological techniques were used to characterize a diencephalic projection from the ventral thalamus to reticulospinal neurons and its role in initiating rhythmic locomotor activity in the spinal cord of adult lampreys (Lampetra fluviatilis). Injection of fluorescein‐coupled dextran amine (FDA) into the rhombencephalic reticular nuclei labeled neurons in the ventral thalamus region on both the ipsilateral side and the contralateral side. Injection of FDA into the ventral thalamus labeled axonal projections in all reticular nuclei, but no direct projections were found to the spinal cord. Extracellular stimulation of the ventral thalamus elicited monosynaptic excitatory postsynaptic potentials (EPSPs), polysynaptic EPSPs, and inhibitory postsynaptic potentials (IPSPs) in reticulospinal neurons in the posterior (prrn) and middle (mrrn) rhombencephalic reticular nuclei. The monosynaptic EPSPs were blocked by the glutamate antagonist kynurenic acid and can be considered glutamatergic. The monosynaptic EPSPs were potentiated (up to 12 minutes) following a brief high‐frequency stimulation. Stimulation of the ventral thalamus induced rhythmic firing of reticulospinal neurons and elicited rhythmic burst activity in the spinal ventral roots. The projections from the ventral thalamus to the reticulospinal neurons in the prrn and mrrn thus provide excitatory inputs to the reticulospinal neurons, which, in turn, can activate the spinal circuits underlying locomotion. Also, the input nuclei to the ventral thalamus were labeled following injection of FDA into this nucleus. Labeled cells were found in the olfactory bulb, pallial areas, striatum, preoptic nucleus, hypothalamus, dorsal thalamus, optic tectum, and dorsal isthmic gray. The ventral thalamus, therefore, receives inputs from several different regions in the brain and controls the level of excitability in reticulospinal neurons. J. Comp. Neurol. 389:603–616, 1997.

Organization of the lamprey striatum – transmitters and projections

The purpose of the present study is to characterize the striatum of the lamprey by immunohistochemical and tracing techniques. Cells immunoreactive for GABA and substance P (SP), and positive for acetylcholinesterase, are present in the lamprey striatum. Immunoreactive (ir) fibers were detected by antisera raised against SP, dopamine, enkephalin and serotonin. These immunoreactive fibers were mainly located in the periventricular neuropil that borders the striatum and in which GABAergic striatal neurons distributed their dendritic arbors. Putative connections between the striatum, the ventral part of the lateral pallium, and the diencephalic motor centers involved in the control of locomotion were studied by using fluorescein-coupled dextran amines (FDA) as a tracer. The striatum projects to the ventral part of the lateral pallium (lpv), where GABA-ir cells and SP-ir fibers were also present. The lpv in turn projects to the ventral thalamus, which has descending connections to the reticulospinal cells involved in the control of locomotion. These results, together with previous findings of histaminergic and neurotensin projections, suggest that the lamprey striatum and its inputs with regard to neurotransmitters/modulators are very similar to those of modem amniotes, including primates, and are thus conserved to a high degree.

AFFERENT AND EFFERENT CONNECTIONS OF THE PARAPINEAL ORGAN IN LAMPREYS : A TRACT TRACING AND IMMUNOCYTOCHEMICAL STUDY

The neural connections of the parapineal organ of two species of lampreys were studied with the fluorescent dye 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI) and with immunocytochemistry. The lamprey parapineal organ consists of a vesicle and a ganglion that are connected to the left habenula. Labeling experiments included the application of DiI to the parapineal organ, left and right fasciculus retroflexus, left habenula, and the left pretectal region. Afferent parapineal fibers run in the left fasciculus retroflexus to the interpeduncular nucleus. The parapineal fibers of this fascicle arose from parapineal ganglion cells, whereas DiI application to the left habenula labeled both neurons of this ganglion and bipolar cells in the parapineal vesicle. Efferent neurons were observed in the left habenula, and bilaterally in the subhippocampal nucleus and the dorsal pretectum. Labeling with DiI also revealed a hippocampal projection.

New and Old Thoughts on the Segmental Organization of the Forebrain in Lampreys

Ten years ago, we published the first detailed prosomeric map of the forebrain in lampreys. In the interim, the prosomeric model has been modified and simplified to better explain numerous data on the expression patterns of regulatory genes, as well as data from chemical, hodological and neuroembryological experiments, mostly in amniote vertebrates. In this report we first review the main historical concepts of lamprey forebrain organization, relating them to either columnar- or segmental-influenced models and explicit or implicit axial references. Next, our previous prosomeric model of the lamprey forebrain is updated, postulating some new hypotheses on the organization of the secondary prosencephalon.

Cholinergic and GABAergic neuronal elements in the pineal organ of lampreys, and tract-tracing observations of differential connections of pinealofugal neurons.

Abstract The putative cholinergic and GABAergic elements of the pineal organ of lampreys were investigated with immunocytochemistry to choline acetyltransferase (ChAT) and γ-aminobutyric acid (GABA), and by acetylcholinesterase (AChE) histochemistry. For comparison we also carried out immunocytochemistry to serotonin (5-HT) and a tract-tracing investigation of the two types of projecting cells, i.e., ganglion cells and long-axon photoreceptors. Most photoreceptors were ChAT-immunoreactive (ChAT-ir) and AChE-positive, while ganglion cells and the pineal tract were ChAT-negative and AChE-negative or only faintly positive. These results strongly suggest the presence of a cholinergic system of photoreceptors in the lamprey pineal organ. GABA-ir fibers that appear to originate from faintly to moderately stained ganglion cells were observed in the pineal stalk. Immunocytochemistry to 5-HT indicated the presence of two types of 5-HT-ir cells, bipolar cells and ganglion-like cells. The connections of the ganglion cells and long-axon photoreceptors were also studied by application of DiI to the pineal stalk in fixed brains or of biotinylated dextran amine (BDA) to one of the main targets of pinealofugal fibers (optic tectum or mesencephalic tegmentum) in isolated brains in vitro. Some long-axon photoreceptors and ganglion cells were labeled from the optic tectum. However, BDA application to the tegmentum exclusively labeled ganglion cells in the pineal organ. These results indicate that the two morphological types of afferent pineal neuron have different projections. No labeled cells were observed in the parapineal organ in BDA experiments, indicating that this organ and the pineal organ are involved in different neural circuits.

Forebrain dopamine depletion impairs motor behavior in lamprey.

The structure of the basal ganglia appears to be conserved throughout vertebrate evolution, with characteristic cellular and transmitter components in each area, and the same types of afferent input. As described in rodents and primates, depletion of the striatal dopamine results in characteristic motor deficits. To explore if this role of the basal ganglia in modulating motor function was present early in vertebrate evolution, we investigated here the effects of striatal dopamine depletion in the lamprey, a cyclostome, which diverged from the main vertebrate line around 560 million years ago. The lamprey striatum contains the same cellular elements as found in mammals, and receives the same types of input, including a prominent dopamine innervation. We show here that MPTP (1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine; 100 mg/kg i.p.), a neurotoxin, depletes forebrain and striatal dopamine levels in lamprey to 15% of control values, and has profound effects on motor performance. Twenty‐four and 48 h after MPTP injection, lampreys demonstrated marked reductions in spontaneous swimming and the duration of each swimming episode. Impairments in the ability to initiate movements were shown by a decreased rate of initiation. Furthermore, the initiation and maintenance of locomotion induced by olfactory mucosa stimulation was severely impaired, as was the coordination of different motor tasks. These deficits were ameliorated by the dopamine agonist apomorphine. The motor deficits arising after striatal dopamine depletion are thus qualitatively similar in cyclostomes and mammals. The role of the dopamine innervation of the striatum thus appears to be conserved throughout vertebrate evolution.

Distal-less-like protein distribution in the larval lamprey forebrain.

A polyclonal antibody against the Drosophila distal-less (DLL) protein, cross-reactive with cognate vertebrate proteins, was employed to map DLL-like expression in the midlarval lamprey forebrain. This work aimed to characterize in detail the separate diencephalic and telencephalic DLL expression domains, in order to test our previous modified definition of the lamprey prethalamus [Pombal and Puelles (1999) J Comp Neurol 414:391-422], adapt our earlier schema of prosomeric subdivisions in the lamprey forebrain to more recent versions of this model [Pombal et al. (2009) Brain Behav Evol 74:7-19] and reexamine the pallio-subpallial regionalization of the lamprey telencephalon. We observed a large-scale conservation of the topologic distribution of the DLL protein, in consonance with patterns of Dlx expression present in other vertebrates studied. Moreover, evidence was obtained of substantial numbers of DLL-positive neurons in the olfactory bulb and the cerebral hemispheres, in a pattern consistent with possible tangential migration out of the subpallium into the overlying pallium, as occurs in mammals, birds, frogs and teleost fishes.