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Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Submitted 12 October 2007; accepted in final form 26 June 2008
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ABSTRACT |
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INTRODUCTION |
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560 million years ago (Kumar and Hedges 1998
), lampreys represent the most ancient vertebrate group that has developed all main parts of the CNS. Because of their critical position in vertebrate evolution and their relative simplicity, they have been used as an experimental model system for the analyses of the cellular bases of vertebrate motor behavior. The lamprey brain stem-spinal cord circuitry responsible for locomotion, posture, and steering has been described in considerable detail (for review, see Grillner et al. 2000) and provides now a solid platform to address questions concerning the supraspinal control of locomotion.
In lampreys, as in other vertebrates, locomotor activity can be initiated by stimulation of the di- or mesencephalic locomotor regions (DLR, MLR). These regions contain neurons that project monosynaptically to reticulospinal neurons that in turn activate the spinal locomotor networks (Brocard and Dubuc 2003; El Manira et al. 1997; Le Ray et al. 2003). The MLR has been investigated in many classes of vertebrates including the lamprey and can be described as a command center that can initiate and control the frequency of locomotor movements (Brudzynski et al. 1986; Garcia-Rill et al. 1985; Shik et al. 1966; Sirota et al. 2000; for review, see Grillner 1981; Grillner et al. 1997; Jordan 1998). The DLR, on the other hand, has received less attention. It corresponds to the area originally (but incorrectly) designated as the subthalamic locomotor region in cats (Grossman 1958; Orlovsky 1970; see Grillner et al. 1997; Jordan 1998) and the lateral hypothalamus in rats (Sinnamon 1993 see also Milner and Mogenson 1988).
The lamprey DLR coincides with a region referred to as the ventral thalamus (El Manira et al. 1997; see also Pombal and Puelles 1999), which contains neurons that project to the lower brain stem reticulospinal nuclei. Moreover, in in vitro brain/spinal cord preparations, electrical stimulation of this region elicits monosynaptic excitatory postsynaptic potentials (EPSPs) and rhythmic firing in reticulospinal neurons as well as rhythmic locomotor-like activity in the ventral roots (El Manira et al. 1997). In the present study, one aim was to investigate in the semi-intact preparation (Sirota et al. 2000) if the rhythmic activity generated by stimulation of the DLR actually produced well-coordinated locomotor movements and if the level of activity could also be controlled from DLR. The semi-intact preparation leaves the body free to move, while the brain stem is exposed but held immobile.
A second goal was to describe the afferent input to DLR, in particular in relation to the input from GABAergic neuronal structures. We have recently shown that both MLR and tectum (controlling eye and orientation movements) both receive GABAergic projections from certain pallial regions (Ménard et al. 2007; Robertson et al. 2006). These areas in turn receive GABAergic projections from the lamprey striatum, and they may therefore correspond to the output nuclei of the lamprey basal ganglia. The striatum of the lamprey has a similar organization to that of mammals with regard to neural elements and afferent input (Pombal et al. 1997a,b). A dopamine denervation produces severe hypokinetic deficits of the same type as in mammals (Ménard et al. 2003, 2004; Grillner et al. 2000; Thompson et al. 2008). In mammals, GABAergic cells from the output nuclei of the basal ganglia control different motor centers, including tectum/superior colliculus and MLR through tonic inhibition at rest (Garcia-Rill 1986; Hikosaka et al. 2000; Mink 1996; Takakusaki et al. 2003; see Grillner et al. 2005). The same motor centers receive tonic GABAergic inhibition in lamprey (Ménard et al. 2007; Robertson et al. 2006). In the present study, we therefore investigate the GABAergic modulation of the lamprey DLR and the forebrain GABAergic projections to the DLR.
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METHODS |
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Physiology
Seventy-five postmetamorphic adult lampreys 37 Petromyzon marinus (length 10–15 cm), 38 Lampetra fluviatilis (length around 30 cm) were used for the physiological experiments. No obvious differences have been found between the two lamprey species with regard to both physiology of the motor system (see Grillner et al. 2008) and the distribution of GABAergic neurons (Robertson et al. 2007) and other transmitter-identified systems.
Surgical procedures
During the surgery, the animals were deeply anesthetized with tricaine methane sulfonate (MS-222; 100 mg/l; Sigma, St. Louis, MO) diluted in fresh water. In semi-intact preparations, the brain was exposed dorsally by removing all the muscles, and connective tissue from the head region to the fourth gill while the rest of the body was kept intact. To discontinue anesthesia, a rostral portion of the telencephalon was removed (the olfactory bulbs and the dorsal half of the lateral pallium). The habenular and posterior commissures as well as the tectum were cut open dorsally to allow access to the DLR. The animals were then transferred to a chamber perfused with cooled (8–12°C) Ringer (which contained, in mM: 138 NaCl, 2.1 KCl, 1.8 CaCl2, 1.2 MgCl2, 4 glucose, and 2 HEPES; pH of 7.4) in which the isolated brain was pinned down but the tail free too move (Fig. 1 A). In in vitro preparations, the brain and the rostral spinal cord were dissected out of the animal and pinned down in a chamber perfused with cooled Ringer.
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In semi-intact preparations, locomotion was monitored with a video camera (Panasonic NV-GS 11, Tokyo, Japan; sampling rate: 25 frame/s) and electromyograms (EMGs). EMGs of the myotomes caudal to the dorsal fin on each side of the body of the lamprey were recorded with pairs of single stranded Teflon-insulated stainless steel wires (0.5 mm ID, 1.125 mm OD; A-M Systems, Carlsborg, WA). The signals were differentially amplified (Differential AC amplifier No. 1700, A-M Systems; 10,000x), filtered (bandwidth: 100 Hz to 5 kHz) and acquired at a sampling rate of 10 kHz through a DigiData 1200 interface (Axon Instruments, Union City, CA) with Clampex 9 software (Axon Instruments). Video frames were captured in AVI format with the software Pinnacle Hollywood FX 8, and the outlines of the body of the lamprey were drawn in Flash MX (Macromedia, Toronto, ON, Canada). In in vitro preparations, fictive locomotion was monitored with electroneurograms (ENGs). ENGs were recorded from the ventral roots with glass suction electrodes filled with Ringer solution and band-pass filtered between 100 and 1,000 Hz. Intracellular recordings of reticulospinal cells were performed with sharp glass micropipettes pulled from thin-walled aluminosilicate glass (Sutter Instrument, Novato, CA) to a final resistance of 80–120 M
, filled with 3 M potassium acetate. The intracellular signals were recorded with an Axoclamp 2A amplifier (Axon Instruments) and amplified 50 times. EMGs and ENGs were analyzed with Datapac software (Run Technologies, Mission Viejo, CA).
Stimulations and drug injections
The habenula, the fasciculus retroflexus, the infundibular recess, and the ventriculus lateralis were used as landmarks to precisely position the stimulating electrode into the DLR (El Manira et al. 1997). The medial pallium, the ventriculus lateralis, and the nucleus preopticus were used as markers to identify the position of the striatum. The DLR was also identified physiologically as the best location in the thalamus in which locomotion could be induced by unilateral electrical or chemical stimulation. The size of the effective region was 100–300 µm in diameter. Injection of a fluorescent tracer into the stimulation site at the end of the experiment was done for six animals to confirm the position of the electrode determined by inspecting 20 µm transversal sections of the brain (Fig. 1B). Electrical stimulation was produced with an isolated pulse stimulator No. 2100 (A-M Systems) connected to tungsten concentric bipolar electrodes (SNEX-100, 100 µm cross-section, impedance: 0.7–1.5 M, epoxylite insulation; Clark Electromedical Instruments, Pangbourne, UK). Chemical stimulation was produced with D-glutamate (5 mM; Sigma) pressure-injected with a Picospritzer II (Parker Hannifin, General Valve Division, Fairfield, NJ) through a glass micropipette lowered into the tissue just below the surface. Ten- to 20-ms pulses at 20 psi were used to inject gabazine hydrobromide (SR 95531, 1 mM; Tocris, Ellisville, MO), or muscimol hydrobromide (1 mM; Sigma) into the brain. All drugs were diluted in Ringer's containing Fast Green to visualize the extent of the injection.
Anatomy
SURGICAL PROCEDURES USED FOR TRACER INJECTIONS. Anatomical experiments were performed on 22 adult lampreys (Lampetra fluviatilis). The animals were kept in a bath perfused with Ringer solution at 8–12°C containing the anesthetic MS222 throughout the procedure and were spinalized at the most rostral level of the spinal cord. The brain was exposed by a minimal opening of the skull and the habenular and posterior commissures as well as the tectum were cut open dorsally to allow access to the DLR. Neurobiotin (Vector, Burlingame, CA; 10–15% in distilled water containing fast green) was injected into the DLR with a glass micropipette connected to a Picospritzer. The landmarks described in the preceding text were used to precisely position the glass micropipette into the DLR. The animals were then left to rest in the bath for 24 h to allow transport of the tracer after which they were perfused through the ascending aorta with 4% formalin, 2% glutaraldehyde, and 14% picric acid in 0.1 M phosphate buffer (PB). The brains were then removed and postfixed for another 24–48 h and cryoprotected in 20% sucrose in PB overnight. Serial transverse sections were cut at 20 µm on a cryostat and collected onto gelatinized slides.
GABA immunohistochemistry and tracer visualization
Sections were preincubated with 1% bovine serum albumin (BSA), 0.3% Triton X-100 in 0.1 M PB for 1 h. The primary antibody, a mouse monoclonal anti-GABA (0.1 µg/ml), was then applied to the sections for 1 day at 4°C. After thorough rinsing in 0.01 M PB saline (PBS), the sections were incubated for 1 h in a mixture of streptavidin conjugated to Cy3 (1:1,000; Jackson Immunoresearch, West Grove, PA) or to Alexa 488 (1:1,000; Molecular Probes, Eugene, OR) for visualization of the Neurobiotin and donkey anti-mouse IgG conjugated to Cy3 (1:800; Jackson Immunoresearch) for visualization of GABA antibodies. Sections were finally thoroughly rinsed in PBS and mounted in glycerol containing 2.5% diazabicyclanooctane (Sigma).
The monoclonal anti-GABA antibody mAb 3A12 was kindly donated by Dr. Peter Streit, Zürich, Switzerland. This antibody was developed after immunization with GABA coupled to BSA and has been well characterized by enzyme-linked immunoabsorpent assays and preabsorption with GABA-BSA (Matute and Streit 1986). Colocalization with mAb 3A12 and a rabbit polyclonal anti-GABA antibody (1:100; No. AB131; Chemicon, Temecula, CA) showed immunoreactivity in the same cells in the lamprey brain. No immunoreactivity was detected when the primary antibody was omitted, and the sections were incubated with only the secondary antibody.
Analysis
Only the brains showing an injection site into the ventral thalamus, rostral to the fasciculus retroflexus, caudal to the nucleus of the postoptic commissure and dorsal to the hypothalamus and a good GABA labeling were included in the analysis (n = 9). Sections from the olfactory bulb caudally to the injection site were analyzed on a Nikon fluorescence microscope, and photomicrographs were taken with a Zeiss Axiocam digital camera (Carl Zeiss AB, Stockholm). Illustrations were prepared in Adobe Photoshop CS1. The nomenclature used is adapted from previous studies (Heier 1948; Nieuwenhuys and Nicholson 1998; Pombal and Puelles 1999; Schober 1964).
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RESULTS |
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The movements that result from DLR activation were investigated in 25 semi-intact preparations, which allow an analysis of the overall coordination along the entire body (Fig. 1A). Electrical stimulation of the DLR evoked rhythmic, symmetric, and well-coordinated swimming movements in 12 of 17 animals. The movements were similar to those of intact animals, which can be described as a wave of lateral displacement of the lamprey body that propagates from head to tail which is symmetric on the right and left side of the body and is of larger amplitude toward the tail (Fig. 1D) (Williams et al. 1989). DLR stimulation did not induced disorganized patterns of motor activity such as C or S shapes (Sirota et al. 2000).
The EMG activity alternated between the left and right side of the body (Fig. 1C). The quality of swimming evoked by stimulation of the DLR was comparable to that induced from MLR in the same animal (n = 10; data not shown). In the cases in which the electrical stimulation failed to induce locomotion, the electrode was moved to other nearby positions within the DLR area delimited by our specific landmarks. In five animals, this was not sufficient to induce swimming, and in some of these animals, a disorganized pattern of movement was instead produced.
To exclude the possibility that the electrical stimulation was producing its effect through activation of fibers of passage, we also stimulated the DLR by a microinjection of D-glutamate that would excite only the cell bodies. In four animals, the swimming movements induced by an electrical stimulation of the DLR (10 pulses, 10 Hz, 20 µA, Fig. 1D1) were compared with those induced by a D-glutamate injection (5 mM, Fig. 1D2). The swimming movements were qualitatively similar as was the frequency (2.3 vs. 2.1 Hz) and the rostrocaudal phase lag from head to tailfin. The lateral displacement was somewhat larger following electrical stimulation. This indicates that neurons within the DLR can induce swimming.
The three graphs in Fig. 1E illustrate how the EMG locomotor burst activity changes with the amplitude of the DLR stimulation (from 7 to 20 µA) during continuous train of stimulation (0.5 Hz). The frequency of swimming increased with the strength of the electrical stimulation of the DLR (Fig. 1E1). Burst amplitude (Fig. 1E3) increased with current intensity, while the burst duration (Fig. 1E2) became correspondingly shorter. This is most likely due to both a recruitment of new motoneurons (previously subthreshold) and an enhanced frequency of already active motoneurons.
DLR stimulation induced fictive locomotion in the in vitro preparation
We analyzed in greater detail the characteristics of the fictive locomotion induced by DLR stimulation in the in vitro preparation (13 of 22 preparations investigated), which is exempt of sensory feedback.
As illustrated in Fig. 2 A1, a train of 10 pulses (10 Hz, 50 µA) could induce a bout of locomotor activity that lasted for several seconds. The longer the stimulus train, the longer the locomotor episode (not illustrated). When a continuous train of stimulation (0.5 Hz, 50 µA) was instead delivered to the DLR, the locomotor episode was maintained for a much longer period (Fig. 2B1; n = 4). Moreover, the continuous train increased the locomotion rate as can be assessed by the comparison of the magnified ENGs in Fig. 2A2 and B2. The graphs of the instantaneous locomotor frequency obtained in each situation show that the rate decreased progressively during a locomotor episode induced by a short train of pulses but that it remained stable at a higher level during a locomotor episode induced by a continuous train of stimulation (Fig. 2C).
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GABAergic input to the DLR modulates swimming in lampreys
The significance of the GABAergic input to the DLR was tested in 10 in vitro and 14 semi-intact preparations by injecting a GABAA agonist (muscimol) and an antagonist (gabazine) into the DLR while monitoring locomotor activity.
For the effect of the GABAA agonist, a control locomotor episode was induced by the injection of D-glutamate (5 mM; Fig. 3 A). The EMG recordings show that swimming occurred 4 s after the onset of the injection and lasted for 14 s. Figure 3B shows the effect of muscimol when injected during a D-glutamate evoked locomotor bout and 38 s later in Fig. 3C where the D-glutamate response had been entirely abolished. However, once the drug was washed out after 15 min, D-glutamate regained its efficacy (Fig. 3D). The results obtained in each animal tested (n = 7) were qualitatively the same. The experiments thus show that cells within DLR have GABAA receptors and that when activated they can modulate locomotor activity.
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To investigate if some of the projecting neurons to the DLR were GABAergic, we combined retrograde tracing, after injection of Neurobiotin into DLR, with GABA immunohistochemistry (9 animals). The distribution of Neurobiotin-labeled cells and tracts were in accordance with previous studies (El Manira et al. 1997; see also Northcutt and Wicht 1997). Figure 5 shows a schematic representation of the distribution. Retrogradely labeled tracts are depicted in yellow and retrogradely labeled cells in green on the left side of the brain sections. From the injection site, some fibers descend toward the hypothalamus, others decussate in the postoptic commissure to form a contralateral bundle of fibers (Fig. 6A), but the majority of the labeled fibers ascend rostrally close to the optic tract (Figs. 5D and 6A). At the transition between the diencephalon and the telencephalon, the fibers bifurcate laterally toward the lateral pallium. Some very fine fibers course through the medial pallium to form the bundle stria medularis (Figs. 5D and 6B). More rostrally, the fibers follow a path under the striatum to reach the lateral pallium (Fig. 5C) and the septum (Fig. 5B). Retrogradely labeled neurons were also examined rostral to the injection site. Many were located bilaterally in the thalamus, the hypothalamus, the nucleus of the postoptic commissure (NCPO), and the preoptic nucleus (PO; Fig. 5, C–E). Others were found mainly ipsilaterally in the pallial and striatal areas. In most animals, a group of cells were labeled in the caudoventral portion of the medial pallium and others in its periventricular cell layer (Figs. 5D and 6B). A more scattered distribution of retrogradelly labeled neurons was observed in the lateral pallium extending to the septum (Fig. 5, B and C). Many cells were concentrated in the area shared with the dorsal pallium and the lateral portion of the striatum and surrounding area (Fig. 5C).
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Striatal contribution to locomotion via DLR
If the basal ganglia output is at the origin of the GABAergic modulation of the DLR, with stimulation of the striatum, the output level of the basal ganglia may be expected to be inhibited and thereby disinhibit DLR and facilitate locomotion. To monitor the effects of the striatal stimulation, we recorded intracellularly from reticulospinal cells in rhombencephalon and the locomotor activity through EMG recordings. Electrical stimulation of the striatum (see METHODS) induced locomotion in 8 of 15 preparations. Figure 7A, inset, shows that the electric pulse induced a polysynaptic EPSP in the reticulospinal cell. A continuous train of electric stimulation (1 Hz, 40 µA), induced after a few seconds a long plateau of depolarization and action potentials (Fig. 7A). Around 10 s after the rising phase of the plateau, a locomotor episode started (Fig. 7B1). The locomotion outlasted the electrical stimulation and continued for the whole duration of the plateau in the reticulospinal cell (n = 4/6). A direct correlation between the plateau depolarization in single reticulospinal cells and the locomotor activity was not clear-cut (Fig. 7B2), although the firing rate of the cells increased from the beginning to the middle of the plateau (4–10 Hz) as did the amplitude of the bursts.
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DISCUSSION |
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It was previously reported that stimulation of the DLR evoked rhythmic alternating ventral root activity in in vitro preparations exempt of sensory feedback (El Manira et al. 1997). In the present study, we demonstrate that the movements resulting from DLR activation represent swimming that is similar to that of the intact animal. Because in the present study active swimming was also induced by D-glutamate in the DLR, we conclude that neurons within DLR, rather than fibers of passage, are responsible for the initiation by electrical stimulation. Moreover, we show that DLR could control the rate of locomotion and the level of the muscle output.
The DLR thus fulfills the criteria required to be described as a locomotor command center. Thus together with MLR (Sirota et al. 2000), it is established that there are two locomotor command centers present in the lamprey as in other vertebrates. Each of them has monosynaptic excitatory connections to reticulospinal neurons in the middle and posterior rhombencepahlic reticular nuclei (El Manira et al. 1997; Sirota et al. 2000), which conveys excitation to the spinal locomotor networks. Each can thus elicit locomotion independently, depending perhaps on the behavioral context. Whether there is a direct interaction between MLR and DLR is not yet known.
GABAergic input to the DLR modulates swimming
In the physiological experiments, GABAA antagonists and agonists were injected into the DLR to assess a possible role for the GABAergic inputs. Whereas GABAA agonists blocked locomotion consistently, GABAA antagonists only occasionally induced locomotion when injected into the DLR. However, gabazine facilitated the induction of locomotion with D-glutamate injections into the DLR and increased EMG amplitudes of ongoing activity. These results with both muscimol and gabazine indicate not only that neurons within DLR have GABAA receptors but also that a blockade of GABAA receptors can facilitate the occurrence of a locomotor response induced by stimulation of DLR and receptor activation can depress it.
A similar study with gabazine on MLR concluded that the GABAergic projections provide a tonic inhibition that once turned off will release the locomotor command (Ménard et al. 2007). The GABAergic input to the DLR, although present, thus appears to be weaker which would explain why the disinhibition produced by gabazine was not sufficient to induce locomotor activity. However, when extra excitation was provided, the presence of a tonic inhibition could be disclosed.
Forebrain GABAergic projections to the DLR
In a previous study, it was shown that neurons in the lateral pallium and other regions project to the DLR (El Manira et al. 1997). In a detailed anatomical report, we recently showed that the neurotransmitter GABA is widely distributed in the adult lamprey brain (Robertson et al. 2007). To identify the GABAergic projections from the forebrain to the DLR, we used the double-labeling technique.
A limited number of double-labeled cells were found in different regions of the forebrain. The available data do not allow a quantitative comparison of the descending GABAergic projections to MLR (Ménard et al. 2007) and DLR, respectively. The finding that the effect of the GABAA receptor antagonist on DLR was less prominent than on MLR could depend on the relative density of GABA projections but also to a variety of other factors. The regions of the forebrain, in which double-labeled GABAergic projection neurons to MLR and DLR were found, are overlapping. They include the medial, dorsal, and lateral pallium.
In anurans, striatal neurons project to an area located just caudal to striatum that is considered to represent the dorsal pallidum (Endepols et al. 2004; Muhlenbrock–Lenter et al. 2005). This area projects to the dorsal thalamus. In amniotes, GABAergic neurons serve as interneurons within pallium and not as output neurons with long axons. We therefore explore the possibility that the long GABAergic projections from specific portions of the pallium to the DLR, identified here, may correspond to the output nuclei of the basal ganglia.
Lateral to the ementia thalami, in the ventrocaudal part of the medial pallium, a discrete group of GABAergic cells were doubled labeled. GABAergic projections to the tectum and the MLR also originate from the same area (Ménard et al. 2007; Robertson et al. 2006). This area is located just caudal to striatum, similar to the condition in anurans. In teleosts, the ementia thalami is thought to be derived from the diencephalon and a region located more laterally is referred to as the entopeduncular complex and is considered to be of telencephalic origin (Braford and Northcutt 1983; Wullimann and Mueller 2004a; Wullimann et al. 1996). The mammalian entopeduncular nucleus is an integral part of the pallidum of the basal ganglia. The anuran anterior entopeduncular nucleus is now homologized with part of the mammalian pallidum but with the teleostean entopeduncular complex the situation is unclear (Wullimann and Mueller 2004b). The homology with the caudoventral portion of the medial pallium in lamprey, and the entopeduncular complex is as yet to be confirmed. It contains, however, GABAergic projecting neurons to the same brain stem structures as in mammals. Projections from striatum to this area have been established (Northcutt and Wicht 1997; Pombal et al. 1997b; Robertson et al. 2007).
Based on neurohistochemical criteria, the GABAergic neurons in the ventral part of the lateral pallium in the lamprey have been proposed to be homologous to the ventral pallidum as described in anamniotes and amniotes (Pombal et al. 1997a; Reiner et al. 1998; Weigle and Northcutt 1999). In the present study, this region was found to have GABAergic projecting neurons to DLR and previously also to MLR (Ménard et al. 2007). The present study also demonstrated a direct GABAergic projection from the area of striatum as previously for MLR (Ménard et al. 2007). Whether striatal GABAergic cells provide direct inhibition to the DLR or if this region belongs to a subpopulation of pallidal neurons that would intermingle with striatal neurons is as yet unknown.
Because the gene Nkx2.1, considered to be important for the development of the pallidum (Sussel et al. 1999), is not expressed in the ventral telencephalon of lamprey embryos (Murakami et al. 2005; Osorio et al. 2005), the presence of a pallidum in lamprey have been questioned. These studies are, however, limited to a few embryonic stages. The input layer of the basal ganglia, the striatum, has qualities that are found in higher vertebrates, which suggests that the general functional organization of the basal ganglia may be similar to that of mammals. The lamprey striatum thus contains spiny neurons immunoreactive to GABA, substance P, enkephaline, and neurons positive for acetylcholinesterase (Pombal et al. 1997a). Patch-clamp recordings show that striatal neurons in lamprey express the hallmarks of mammalian striatal neurons such as inward rectifier and IH channels (Ericsson et al. 2007). The striatum receives input from a variety of structures (thalamus, pallium), including a strong dopaminergic, serotoninegic, and histamininergic input from neurons of the same origin as in mammals (Pombal et al. 1997a,b). The striatum also sends axons to medial pallium (Northcutt and Wicht 1997), and the lateral pallium (Pombal et al. 1997a). Dopamine depletion results in the same type of hypokinetic symptoms as in mammals, which are counteracted by administration of the dopamine agonist apomorphine (Thompson et al. 2008).
In addition to the double-labeled GABAergic projection neurons, there are a number of other areas which are retrogradely labeled from DLR (El Manira et al. 1997), which have also been identified here. They may represent input to DLR, but the possibility that some fibers of passage have been labeled must also be considered. Even though our injections were aimed at the periventricular cell layer of the ventral thalamus, some axons may have been interrupted en route to their final target. Many fibers in the medial pallium were labeled, and it is considered a main telencephalic target of the thalamus (Northcutt and Wicht 1997; Polenova and Vesselkin 1993). Mitral cells of the medial olfactory bulb were retrogradely labeled (El Manira et al. 1997). Their axons course medially through the thalamus to continue to the caudal hypothalamus (Northcutt and Puzdrowski 1988; Northcutt and Wicht 1997). Other descending tracts originating from different pallial areas to the habenulae, pretectum, tectum, or midbrain could also have been labeled by the injection (Northcutt and Wicht 1997; Polenova and Vesselkin 1993; for review, see Nieuwenhuys and Nicholson 1998).
DLR relation to the striatum
Given that both the structure and function of the striatum appear to be conserved from cyclostomes to mammals, it would appear likely that striatum acts via the putative pallidal structures with GABAergic projection neurons that target the DLR. In the present study, electrical stimulation of the striatum induced long plateau potentials in reticulospinal cells associated with locomotor episodes. Moreover, activation or inhibition of the DLR, respectively, facilitated or inhibited the effects of the striatal stimulation, which implies that DLR contributed to the initiation of locomotion. This suggests that the activation of the striatum inhibits the GABAergic projections to the DLR which in turn will lead to a disinhibition of DLR that can then excite the reticulospinal cells which will in turn initiate locomotion. These results indicate thus that DLR is of importance for the effects elicited from striatum, but some of these effects may in addition be exerted via MLR. In addition, the stimulation of the striatum seems to be more effective in the disinhibition the DLR than an injection of the GABAA antagonist into the DLR because we were able to get locomotor activity on stimulation. This could be due to the electrical stimulation used at the level of the striatum that could have activated some fibers of passage or nearby structures. However, an injection of D-glutamate into the striatum also succeeds in inducing locomotion (personal observation).
A further understanding of the interaction between striatum and the presumed pallidal structures will, however, require detailed studies of the synaptic action exerted by striatum on the pallidal GABAergic neurons, and recordings of their pattern of activity at rest and during initiation of motor behavior.
Concluding remarks
DLR and MLR can both serve as command regions for locomotion. They both contain neurons that bilaterally project to reticulospinal neurons in the middle and posterior reticulospinal nuclei that will turn on the spinal networks that generate the locomotor movements. DLR and MLR appear both to be subject to a GABAergic tonic inhibition under resting conditions because GABAA antagonists will trigger or enhance the occurrence of locomotor activity. Both structures receive long GABAergic projections from a group of cells in the caudoventral part of the medial pallium near eminentia thalami and from another group in the ventrolateral part of the lateral pallium. These GABAergic structures may correspond to the basal ganglia output nuclei in other vertebrates, and the motor effects elicited by stimulation of the striatum may be channeled via these nuclei.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Grillner, Nobel Institute for Neurophysiology, Dept. of Neuroscience, Karolinska Institutet, Retzius vag 8, SE-171 77 Stockholm, Sweden (E-mail: Sten.Grillner{at}ki.se)
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and 
, respectively). D: graph showing the effect of frequency changes in the continuous train stimulation of the DLR on the frequency of fictive swimming induced.
). B: the swimming evoked by D-glutamate (2 1st 
in A). Note the different time scales in B1 and B2.