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Muscarinic Receptor Activation Elicits Sustained, Recurring Depolarizations in Reticulospinal Neurons Address for reprint requests and other correspondence: S. Alford, Univ. of Illinois at Chicago, Dept. of Biological Sciences (MC 067), 840 West Taylor St., Rm. 4285, Chicago, IL 60607 (E-mail: sta/at/uic.edu)  The publisher's final edited version of this article is available free at J Neurophysiol. | |||||||||||||||||||
Abstract In lampreys, brain stem reticulospinal (RS) neurons constitute the main descending input to the spinal cord and activate the spinal locomotor central pattern generators. Cholinergic nicotinic inputs activate RS neurons, and consequently, induce locomotion. Cholinergic muscarinic agonists also induce locomotion when applied to the brain stem of birds. This study examined whether bath applications of muscarinic agonists could activate RS neurons and initiate motor output in lampreys. Bath applications of 25 μM muscarine elicited sustained, recurring depolarizations (mean duration of 5.0 ± 0.5 s recurring with a mean period of 55.5 ± 10.3 s) in intracellularly recorded rhombencephalic RS neurons. Calcium imaging experiments revealed that muscarine induced oscillations in calcium levels that occurred synchronously within the RS neuron population. Bath application of TTX abolished the muscarine effect, suggesting the sustained depolarizations in RS neurons are driven by other neurons. A series of lesion experiments suggested the caudal half of the rhombencephalon was necessary. Microinjections of muscarine (75 μM) or the muscarinic receptor (mAchR) antagonist atropine (1 mM) lateral to the rostral pole of the posterior rhombencephalic reticular nucleus induced or prevented, respectively, the muscarinic RS neuron response. Cells immunoreactive for muscarinic receptors were found in this region and could mediate this response. Bath application of glutamatergic antagonists (6-cyano-7-nitroquinoxaline-2,3-dione/D-2-amino-5-phosphonovaleric acid) abolished the muscarine effect, suggesting that glutamatergic transmission is needed for the effect. Ventral root recordings showed spinal motor output coincides with RS neuron sustained depolarizations. We propose that unilateral mAchR activation on specific cells in the caudal rhombencephalon activates a circuit that generates synchronous sustained, recurring depolarizations in bilateral populations of RS neurons. | |||||||||||||||||||
In lampreys, brain stem reticulospinal (RS) neurons initiate, maintain, and modulate motor output (Buchanan and Grillner 1987; Deliagina et al. 2002; Ohta and Grillner 1989; for a review see Brodin et al. 1988). They provide the main descending input to the spinal cord (Brodin et al. 1988; Nieuwenhuys 1977; Rovainen 1979) and directly activate the spinal central pattern generators (CPGs) that mediate locomotion (Buchanan and Cohen 1982; Rovainen 1974; for a recent review, see Grillner 2003). RS neurons receive sensory inputs from several modalities (Alford and Dubuc 1993; Deliagina et al. 1993; Finger and Rovainen 1982; Orlovsky et al. 1992; Viana Di Prisco et al. 1995; Zompa and Dubuc 1996). They also receive inputs from the spinal locomotor networks (Dubuc and Grillner 1989; Einum and Buchanan 2004; Vinay and Grillner 1992) and from the supraspinal locomotor control centers in the mesencephalon (Brocard and Dubuc 2003; Le Ray et al. 2003) and diencephalon (El Manira et al. 1997). In addition to glutamatergic inputs from the mesencephalic locomotor region (MLR), RS neurons receive cholinergic nicotinic excitation (Le Ray et al. 2003). In birds (ducks and geese), locomotor behavior is induced by injections of carbachol, a nonspecific cholinergic agonist, into the brain stem, an effect that is blocked by the muscarinic acetylcholine receptor (mAchR) antagonist, atropine (Sholomenko et al. 1991). Muscarinic acetylcholine receptors modulate numerous physiological functions in the central and peripheral nervous system. The mAchR family (M1–M5) belongs to the plasma membrane–bound G protein– coupled receptor superfamily (Caulfield and Birdsall 1998; Wess 1996). Regional localization, pre- or postsynaptic position, as well as downstream signal transduction machinery, influence the physiological response to mAchR activation (Felder et al. 2000). In lampreys, muscarinic agonists were recently shown to depress sensory inputs to RS neurons (Le Ray et al. 2004). No excitatory effects were observed in RS neurons in response to the muscarinic agonists. However, because the drugs were only locally applied over the RS neurons or the sensory relay cells, the possibility that mAchR activation could indirectly activate RS neurons through actions in other parts of the brain stem was not studied. We examined the effects of bath applications of muscarinic agonists on the rhombencephalon and found that sustained, recurring depolarizations were systematically elicited in RS neurons. The depolarizations did not result from a direct effect on the RS neurons but most likely from the activation of muscarinoceptive cells in the caudal half of the rhombencephalon. Local, unilateral applications of muscarine induced bilateral populations of RS neurons to activate synchronously. Also, motor output recorded from ventral roots coincided with the muscarine induced depolarizations in RS neurons. | |||||||||||||||||||
Animals Larval and young adult lampreys (Petromyzon marinus) were collected in southern Québec from streams entering Lake Champlain. Some animals were purchased from ACME Lamprey Co. (Harrison, ME). Adult lampreys used in immunohistochemical experiments were obtained from the Ste. Mary’s River in Ontario or the Great Chazy River in New York State. All experimental procedures conformed to The Association for Assessment and Accreditation of Laboratory Animal Care–International (AAALAC) and the Canadian Council on Animal Care (CCAC). They were also approved by the Animal Care Committees of UIC, Université de Montréal, and Université du Québec à Montréal.In vitro isolated rhombencephalon preparation Experiments were performed on the isolated rhombencephalon of larval (n = 88) or young adult (n = 4) lampreys. The animals were anesthetized with tricaine methanesulfonate (MS-222; 100 mg/l; Sigma Chemical, St. Louis, MO), decapitated, and dissected in a cold saline solution (Ringer) of the following composition (in mM): 100 NaCl, 2.1 KCl, 2.6 CaCl2, 1.8 MgCl2, 4 glucose, and 26 HEPES, adjusted to a pH of 7.60. The skin, muscle, and viscera were removed. The dorsal surface of the cranium was opened, and the nasal tissue rostral to the isthmus and caudal to the second/third spinal segments was removed. Although technically the preparation included a few spinal segments, it was referred to as the “isolated rhombencephalon” for simplicity. It is noteworthy that results were the same whether the few rostral spinal segments were kept or removed (lesion at the level of the obex; see RESULTS). A midsagittal, dorsal transection was made at the level of the isthmus and allowed spreading the alar plates on the lateral sides of the fourth ventricle. This provided open access to the RS neurons. The procedure of transecting the isthmus is represented by the schematic in Fig. 1A. The brain case, containing the isolated rhombencephalon preparation, was pinned down to the Sylgard bottom of a cooled, 5-ml chamber (Fig. 1A). The recording chamber was continually superfused with cold oxygenated Ringer (8 –10°C).
In vitro rhombencephalon–spinal cord preparation In contrast to the previous preparation, 15–25 segments of the spinal cord were kept attached in three preparations. This rhombencephalon–spinal cord preparation was pinned down and maintained in a chamber perfused with Ringer solution as previously described. Ventral root (VR) activity was recorded extracellularly on both sides using glass suction electrodes filled with the Ringer solution.Electrophysiology Intracellular recordings were made from RS neurons in the middle (MRRN, n = 63) and in the posterior (PRRN, n = 7) rhombencephalic reticular nuclei, using sharp glass microelectrodes filled with 3 M potassium acetate (80 –120 MΩ). The signals were amplified by an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA; sampling rate: 2–10 kHz). Only RS neurons with a stable membrane potential, held for 15 min after impalement, less than −65 mV were included in the study. Ventral root recordings (n = 3) were performed with glass extracellular suction electrodes filled with Ringer solution. Ventral root recordings were amplified with a differential AC amplifier (A-M Systems, Carlsberg, WA).Drugs All drugs were dissolved to their final concentration in Ringer solution containing the inactive dye fast green. Muscarine chloride (muscarine, 25 μM; Sigma-Aldrich, St. Louis, MO), pilocarpine (25–75 μM; Sigma-Aldrich), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 μM; Tocris, Ellisville, MO), D-2-amino-5-phosphonovaleric acid (AP5; 200 μM; Tocris), TTX (1 μM; Calbiochem) were bath applied at the indicated concentrations per experiment. Drugs were bath applied for long enough to reach full concentration in the perfusion chamber (range, 1.5–3 min). Full concentration was considered the point at which the fast green color reached a steady state. Acetylcholine (500 μM; Sigma-Aldrich), muscarine chloride (75 μM), and atropine (1 mM; Sigma-Aldrich) were also pressure-ejected through a glass micropipette with a Picospritzer (General Valve, Fairfield, NJ).For the microinjection experiments, the inactive dye fast green was added to the drug solution to monitor the extent of the application. The injection micropipette was positioned on the surface of the tissue and injection parameters were set so a fast green stain of ~100 μm diameter would be visible on the surface of the tissue right after injection. Control injections of fast green dissolved in Ringer alone did not initiate any response in intracellularly recorded RS neurons. Data acquisition and analysis Electrophysiological data were acquired through a Digidata 1322A AD converter with Clampex 8.0 software (Axon Instruments). Intracellular signals were analyzed with Clampfit 8.0 software. The duration and the period of recurrence of individual depolarizations were analyzed for the first 10 min after the onset of the muscarine response (Fig. 1, B2 and C1,2). Depolarization duration was defined as the time difference between the beginning of a single depolarization and the repolarization to baseline level (Fig. 1 B2). The cycle period was defined as the time between the onsets of two consecutive depolarizations (Fig. 1 B2). All means are expressed ± SE. The same parameters were used in the analysis of the calcium imaging data.Labeling of neurons with Ca2+-sensitive dyes Retrograde labeling of neurons with dextran-amine-conjugates of calcium-sensitive dyes has proven to be very effective to examine the activation of populations of neurons in the lamprey CNS (McClellan et al. 1994; Schwartz and Alford 2000; Viana Di Prisco et al. 2000, 1997). Changes in intracellular calcium concentration can be measured in response to various cellular activities, including single pre-synaptic action potentials, synaptic stimulation, and oscillations during rhythmic activity. Two techniques were used for loading dye into RS neurons. Both techniques required the initial dissection as described above. In one method, the spinal cord was transversely cut at segmental level 1 or 2, and calcium green-dextran (10,000 MW; Molecular Probes, Eugene, OR) crystals were immediately placed directly in the lesion. This preparation was perfused in the dark with cold oxygenated Ringer solution, usually for 48 h (range, 24 – 48 h), to allow in vitro retrograde transport of the dye in RS neurons (Viana Di Prisco et al. 1997). The preparation was transferred and pinned down to the bottom of a small chamber for drug application and imaging. Cold, oxygenated Ringer solution was perfused throughout the experiment, which normally lasted 3– 6 h. Dye loading was also accomplished using a glass pipette tapered to the spinal cord diameter filled with 5 mM calcium green-dextran into which the freshly cut end of the spinal cord (the end continuous with the brain stem) was suction inserted. This technique was previously described in detail (Schwartz and Alford 1998). The two filling methods yielded comparable levels of labeling, with the former generally filling a slightly larger number of RS neurons.Imaging Dye-loaded RS neurons were imaged during bath application of 25 μm muscarine using a Nikon epifluorescence microscope and recorded with an intensified CCD camera (Hamamatsu C2400, Bridge-water, NJ; neutral density filter at 50%) at a rate of one 10- to 20-ms exposure per 5 s. Meta Imaging software was used to acquire and analyze the data. Calcium responses were expressed as relative changes in fluorescence ΔF/F.Immunohistochemistry Immunohistochemical experiments were carried out to determine the location of cells displaying immunoreactivity for mAchR in the rhombencephalon of lampreys using the M35 monoclonal primary antibody. Brains from 11 adult sea lampreys (P. marinus) were used in this part of the study. Adult lampreys were chosen to carry out the immunohistochemical experiments because they tend to show more fully differentiated morphological characteristics (i.e., dendritic arbors) than larval animals. The brains were exposed dorsally and immersed in a solution of 2% paraformaldehyde/0.2% picric acid in phosphate-buffered saline [PBS: phosphate buffer 0.1 M, pH 7.4 (PB), + 0.9% sodium chloride] for 6 h at 4°C. They were transferred in 30% sucrose in PB overnight. For each brain, serial cryostat sections 25 μm thick were collected on gelatinized microscope slides and allowed to dry overnight on a warming plate at 37°C. Every 10th section was collected on a separate slide for negative controls.All subsequent steps were carried out at room temperature unless otherwise specified. Sections were washed three times in PBS, and endogenous peroxidase activity was blocked by incubating the sections for 10 min in PBS containing 0.3% H2O2 and 0.1% sodium azide (Li et al. 1987). They were washed again three times in PBS and preincubated in PBS containing 5% normal rabbit serum and 0.4% Triton X-100 for 1 h, followed by an incubation with the M35 monoclonal primary antibody (Argene, N. Massapequa, NY) diluted 1:200 in PBS containing 1% normal rabbit serum and 0.1% Triton X-100 for 20 h at 4°C. This step was followed by three washes in PBS and incubation with the secondary antibody (biotinylated rabbit anti-mouse, Chemicon, Temecula, CA) diluted 1:100 in PBS containing 1% normal rabbit serum and 0.1% Triton X-100 for 1 h. The sections were washed three times in PBS, reacted with ABC Elite Standard kit (Vector, Burlington, Ontario, Canada) for 1 h, and washed again three times in PBS. They were reacted for 5 min with a solution of 0.05% diaminobenzidine in 0.05 M Tris-HCl buffered saline (pH 7.6). The sections were washed three times with PBS, dehydrated, cleared in xylene, and coverslipped with Entellan (VWR International, Montreal, Quebec, Canada). The same procedure was followed for negative controls except that the primary antibody was omitted. Photomicrographs were taken with a DXM1200 digital camera mounted on a E600 microscope (Nikon, Mississauga, Ontario, Canada). Western blot The ability of the M35 antibody to bind lamprey mAchRs was confirmed using SDS-PAGE Western blot analysis (n = 2). Young adult lamprey or rat (22–25 days old) brain was homogenized in cell lysis buffer (in mM: 5 Tris-HCl, 2 EDTA, 0.0001 aprotinin, leupeptin, antipain, and 10 μg/ml phenylmethylsulfonyl flouride) and centrifuged at 500g for 5 min at 4°C. The supernatant was kept and centrifuged again at 4,300g for 20 min at 4°C. The precipitate was kept and diluted in membrane buffer and centrifuged again at 4,300g for 20 min at 4°C. Membrane proteins were quantified in relation to a known bovine serum albumin (BSA) curve. Samples were diluted in buffer (%: 5 β-mercaptoethanol, 2.5 SDS, 10 glycerol; 100 mM Tris, pH 6.8; 1.4 mM bromophenol blue), boiled, loaded (20 μg per lane), and separated on a 7.5% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (100 V, 2 h), and nonspecific binding was blocked in 5% milk diluted in Tris-buffered saline with 0.1% Tween (1 h, 37°C). Muscarinic receptors were tagged with the anti-muscarinic receptor clone M35 (1:1,000, overnight, 4°C) and revealed with horseradish peroxidase– conjugated goat anti-mouse (1:5,000, 60 min, 20°C; Chemicon). Membranes were incubated with chemiluminescent substrate (Western Lightening Chemiluminescence, Perkin Elmer) and exposed to Kodak Biomax film. | |||||||||||||||||||
RS neuron response to bath-applied muscarine Bath application of 25 μM muscarine induced sustained, recurring depolarizations in intracellularly recorded RS neurons (Fig. 1B1). This occurred in 129 of 134 bath applications (n = 80 preparations, 76 larvae, 4 young adults). Of the other five applications, all in larval preparations, muscarine increased synaptic activity in three and failed to induce recordable changes from baseline in the other two. Efforts to induce this effect using pilocarpine proved much less reliable. Bath application of various concentrations of pilocarpine (25–75 μM) only elicited the sustained, recurring depolarizations in 5 of 17 applications (n = 9 preparations). Of the other 12 cases, pilocarpine induced an increase in synaptic activity in four applications, induced a single sustained depolarization in two applications, or had no effect in the other six applications. Although periods of muscarine application ranged from 1 to 20 min, the effects outlasted the drug application and persisted from between 30 and 90 min after washout. Figure 1B2 shows the expansion of the time axis from Fig. 1B1 and indicates two typical RS neuron depolarizations induced by bath-applied muscarine. Analysis of the first 10 min after onset of a response to a muscarine application in seven different preparations (1 cell recorded per preparation), shown across all seven responses, showed the mean period between recurring depolarizations was 55.5 ± 10.3 s, with a range from 14.8 to 214.2 s, and the mean depolarization duration was 5.0 ± 0.5 s, with a range from 2.6 to 9.1 s. Figure 1, C1 and C2, depicts the range of means generated from each experiment for period and depolarization duration, respectively. These data indicate there is a great deal of variability when comparing the response of a single recorded RS neuron in one preparation to the response of a single recorded RS neuron in other preparations. However, there is also significant variability when comparing the response of a singe recorded RS neuron to repeated applications of muscarine in the same preparation (data not shown). That is, consecutive bath applications of muscarine can induce quite different responses, in regard to depolarization duration and period, in the same recorded MRRN RS neuron (data not shown). We compared the responses of RS neurons in the presence of bath-applied 25 μM muscarine using paired intracellular recordings from bilateral RS neurons in the MRRN. RS neurons on both sides exhibited equivalent responses with simultaneous onset, depolarization duration, and period of recurrence (Fig. 2, A and B; n = 4). All four of these experiments consistently showed simultaneous onset, depolarization duration, and period of recurrence of the activity recorded in the paired MRRN RS neurons. Depolarization amplitude, however, varied between the paired recorded RS neurons. To examine if the effects occurred in large populations of RS neurons, a calcium-imaging technique was applied. Figure 3A depicts a raw image of the calcium green retrogradely labeled RS neuron population in the MRRN. Monitoring the intracellular calcium concentration of these neurons during bath application of 25 μM muscarine consistently revealed synchronized oscillations in intracellular calcium concentration among activated neurons (Fig. 3B; n = 6; mean period = 20.6 ± 4.27 s). However, there was some variability in the onset of these oscillations among RS neurons. In the example shown, and typical of all imaged responses, some RS neurons of the MRRN exhibited a sustained calcium plateau (Fig. 3, neurons a–d) on which oscillations occurred, whereas other RS neurons displayed transient oscillations (Fig. 3, neurons e and f). However, in each muscarine application in all six preparations used, once activity began, RS neurons consistently showed synchronized oscillations in intracellular calcium concentration. The range of the periods of the oscillations analyzed (12.4–123.6 s) was similar to the range of the periods analyzed for the recurring sustained depolarizations seen in the electrophysiological recordings of the MRRN response to bath-applied muscarine (range = 14.8–214.2 s).
Similar calcium-imaging experiments focused on calcium green dextran–labeled RS neurons in the PRRN and yielded comparable results. The intracellular calcium concentration during bath application of 25 μM muscarine oscillated with a common periodicity among activated PRRN neurons (Fig. 4, A and B; n = 5; mean period = 23.9 ± 4.03 s). Intracellularly recorded PRRN neurons also showed an analogous response to bath application of 25 μM muscarine as intracellularly recorded MRRN neurons (Fig. 4, C and D; n = 4; mean period = 56.3 ± 24.2 s). The range of periodicity of the intracellular calcium concentration oscillations (range = 16.1–95.6 s) compared with the range of periodicity (range = 16.6–107.7 s) established through intracellular recordings of PRRN neurons.
Localizing the muscarine effect Bath perfusion of 1 μM TTX, before and during 25 μM muscarine perfusion, abolished the response to muscarine in intracellularly recorded MRRN neurons (Fig. 5, A and B; n = 4) and PRRN neurons (data not shown, n = 3). The presence of TTX also consistently abolished the synchronized oscillations in intracellular calcium concentration of MRRN RS neurons induced by muscarine (Fig. 5C, n = 3). Therefore muscarine does not directly excite the RS neurons. To begin to understand the circuitry involved in the muscarine response, we successively lesioned the brain caudal to rostral to establish the minimal region needed to elicit the sustained, recurring depolarizations. No difference in RS neuron response to muscarine occurred between preparations containing regions rostral to the rhombencephalon compared with the isolated rhombencephalon preparation. The isolated rhombencephalon preparation contained the rhombencephalon and two or three of the rostral spinal segments. The RS neuron response to muscarine persisted after a transection was made at the level of the obex that removed the rostral spinal segments (data not shown, n = 3). Only a lesion made between the MRRN and the PRRN reliably prevented muscarine from eliciting a response in intracellularly recorded RS neurons in the MRRN (Fig. 6, A and B; n = 6). These results suggested that a region caudal to the MRRN might mediate this RS neuron response to muscarine. To test this hypothesis, we performed unilateral local, pressure microinjections of 75 μM muscarine in various locations around the isolated rhombencephalon while intracellularly recording an RS neuron in the MRRN. Unilateral, local injections of muscarine produced a rapid, reliable MRRN response when applied to a region lateral to the rostral pole of the PRRN either ipsilateral or contralateral to the recorded RS neuron in the MRRN (Fig. 7, A1 and A2, traces 4 and 5; n = 4). Unilateral injections of acetylcholine (Ach, 500 μM) in this same region induced sustained, recurring depolarizations in recorded MRRN RS neurons (Fig. 7C; n = 4). Analysis of the first two sustained depolarizations after the onset of the response to Ach injection shows that, of the four preparations measured, the mean period of the oscillations was 55.8 ± 16.3 s, with a range from 30.1 to 111.5 s, and the mean depolarization duration was 39.8 ± 14.0 s, with a range from 16.1 to 87.6 s. These values are not statistically different (P > 0.05) than those measured for experiments in which muscarine was injected (mean period = 34.7 ± 6.1 s, range = 20.4 – 49.8 s; mean depolarization duration = 19.8 ± 1.7 s, range ± 15.6 –22.8 s). The lack of statistical difference between the effects of muscarine and Ach injections reflects the striking variability of the depolarization duration and period of each response to the drug. Injections of muscarine made in other regions of the rhombencephalon predominantly did not produce any response (Fig. 7A2, traces 1–3) or rarely produced a weak (few sustained depolarizations), delayed response (initial response time on the order of a few minutes; data not shown). To further probe this region, we attempted to block the response by microinjecting the mAchR antagonist atropine (1 mM) in various regions around the rhombencephalon immediately before bath application of 25 μM muscarine. The following results were consistent in all three preparations tested. Bilateral, local applications of atropine blocked responses to bath applications of muscarine when applied over the same bilateral regions as the muscarine pressure injection experiments. (Fig. 7, B2 and B3; n = 3). On the other hand, unilateral injections of atropine over this region did not block the response (data not shown). Bilateral atropine applications made more rostrally within the rhombencephalon such as lateral to the MRRN or along the midline at the level of the MRRN did not block the muscarine effect. Also, more caudal injections in the rhombencephalon within and lateral to the caudal PRRN did not have any effect (data not shown).
We used an mAchR antibody (M35), which binds all mAchR subtypes (Carsi-Gabrenas et al. 1997; van der Zee and Luiten 1999), to perform immunohistochemistry on the lamprey rhombencephalon to assay whether there are mAchR-immunoreactive cells in this muscarine responsive region. The specificity of the antibody was assessed using the SDS-PAGE Western blot method to compare reactivity between lamprey and rat whole brain membrane protein extracts. The antibody reacted with a major band at 104 kDa and a minor band at 51 kDa in lamprey and reacted with a major band at 70 kDa and minor bands between 70 and 86 kDa and between 35 and 51 kDa in rats (Fig. 8D; n = 2). Cells displaying mAchR immunoreactivity were located at different locations in the rhombencephalon. Some of the RS neurons within the PRRN and MRRN were labeled with the antibody. Notably, transverse sections at the level of the rostral PRRN and at the level between the MRRN and PRRN revealed a cluster of mAchR-positive cells lateral to the PRRN, as well as cells ventral to the IX and X motor nuclei (Fig. 8, B1 and C1). These cells varied in size from small to medium (Fig. 8, B2 and C2). Muscarinic receptor immunoreactivity was also clearly apparent on motoneurons of IX and X, as well as on RS neurons (Fig. 8, B1 and C1; n = 6).
To test whether glutamatergic transmission contributed to the muscarine-induced sustained, recurring RS neuron depolarizations, intracellular recordings from RS neurons in the MRRN were performed during bath applications of 25 μM muscarine in addition to adding glutamatergic antagonists. The isolated rhombencephalon was perfused with 20 μM CNQX and 200 μM AP5 to block AMPA and N-methyl-D-aspartate (NMDA) receptors, respectively, for ≥45 min before and during bath application of muscarine. The glutamatergic antagonists reliably prevented a muscarine-induced response in the recorded RS neuron (Fig. 9, A and B; n = 3). This indicated that glutamatergic transmission contributes to the RS neuron response to mAchR activation in this network.
Motor behavior Reticulospinal neurons activate spinal CPGs to elicit motor output. To study the response of these networks to muscarinic activation of RS neurons, we combined intracellular recordings of RS neurons with bilateral recordings of VRs (Fig. 10A). Bouts of rhythmic VR activity coincided with the sustained depolarization in the recorded RS neuron in the MRRN (Fig. 10B1; n = 3). Expansion of the time axis of the gray boxed region from Fig. 10B1 revealed clear alternations of activity between VRs on both sides of the spinal cord during a single sustained RS neuron depolarization (Fig. 10B2). The RS neuron sustained depolarizations lasted between 4.5 and 9 s. The alternating bursts in VRs on opposite sides of the spinal cord lasted 0.5–1.5 s, which is compatible with lamprey swimming.
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Results from this study reveal that perfusion of the lamprey rhombencephalon with muscarine induces sustained, recurring depolarizations in RS neurons. Also, muscarine consistently induced synchronous, recurring activation of large numbers of RS neurons. These effects are prevented by blocking synaptic transmission with TTX, suggesting common prereticular inputs to RS neurons. Because consecutive bath applications of muscarine can induce varying responses in the same recorded MRRN RS neuron, the baseline excitability of the preparation could contribute significantly to the character of the muscarine response. Cells immunoreactive for mAchR located in the caudal half of the rhombencephalon are likely candidates for providing such excitatory input to RS neurons. In addition, muscarine-induced bouts of rhythmic spinal motor activity that coincided with each RS neuron sustained depolarization. Circuitry Lesions were performed to establish the minimal circuitry needed to induce sustained depolarizations under muscarine. They revealed that the isolated rhombencephalon was sufficient to elicit the activity. Subsequent lesions revealed that a rhombencephalic region caudal to the MRRN was necessary for muscarine to induce the RS neuron depolarizations in the MRRN. Also, immunohistochemistry experiments revealed the presence of mAchR immunoreactive cells located caudal to the MRRN in the lamprey rhombencephalon. Because TTX blocked the muscarine effect, the activation of mAchRs on RS neurons is unlikely to be responsible for the sustained, recurring depolarizations. This is supported by the lack of effect of bath applied muscarine on RS neuron membrane potential and intracellular calcium concentration in the presence of TTX. In the previous study where the effect of muscarinic agents was examined in the brain stem of lampreys (Le Ray et al. 2004), the authors only examined the effects of local applications of muscarinic agonists onto a recorded RS neuron or on muscarinoceptive cells in a sensory relay nucleus in the lateral, rostral half of the rhombencephalon. These local applications of muscarine depressed sensory-evoked synaptic inputs to RS neurons. Also, when they were applied locally to the RS neurons, there was no effect on the resting membrane potential. This suggests that the effects of RS neuron mAchR activation may be more evident in relation to the activity of RS neuron inputs. Relative to the present study, it suggests that the mAchR-mediated excitatory drive occurs at a prereticular level and not directly on RS neurons. Furthermore, although inputs to RS neurons may be depressed during the bath application of muscarine, it is the prominent excitatory drive of the muscarinoceptive region in the caudal rhombencephalon that is revealed by the sustained, recurring depolarizations in RS neurons.Given that the rhombencephalon caudal to the MRRN was needed for muscarine to induce the sustained depolarizations in RS neurons, local microinjections of muscarine were made in the caudal rhombencephalon to further probe the region. Injections in an area lateral to the rostral PRRN reliably induced the characteristic sustained, recurring depolarizations in the MRRN RS neurons. Injections made lateral to the MRRN, lateral to the caudal half of the PRRN, or within the PRRN did not produce any response, or more rarely, produced a weak delayed response suggestive of some drug diffusion. Unilateral injections of muscarine in this region lateral to the rostral PRRN either ipsilateral or contralateral to the recorded MRRN RS neurons proved sufficient to induce the response. This suggests midline crossing within this network most likely prereticulospinal, because no connections between bilateral RS neurons have been previously established (Dubuc et al. 1993). The case for a midline crossing is further supported by the need to make bilateral, as opposed to unilateral, injections of atropine to block the effect during bath application of muscarine. The identity of the cells that directly respond to muscarine is not yet determined, but the mAchR immunoreactive cells located lateral to the rostral pole of the PRRN are strong candidates. These cells could in turn project bilaterally to populations of RS neurons in the MRRN and the PRRN. Anatomical experiments are underway to test this hypothesis. The M35 antibody (Andre et al. 1984) binding to mAChRs has been studied in a variety of species (invertebrate and vertebrate), tissues, and cell types, and to our knowledge, no species are reported in which the M35 antibody is incapable of binding to mAChRs (Carsi-Gabrenas et al. 1997; van der Zee and Luiten 1999). The evolutionary conservation of muscarinic receptor structure has been shown using different methods (Hwang et al. 1999; Venter 1983; Venter et al. 1984). M35 binds to all five subtypes of mAchR (Carsi-Gabrenas et al. 1997). The M35 antibody has also been shown to induce agonist-like activity of mAchRs (Leiber and Harbon 1982). There are no data to suggest that M35 reacts with proteins other than mAchRs. In addition, there are studies that suggest the M35 antibody does not bind to proteins other than mAchR. One study in prenatal mouse embryo compared the distribution of labeling between autoradiography using a mAchR-specific ligand to immunohistochemistry using the M35 antibody (Lammerding-Koppel et al. 1995). Both techniques showed the same pattern of localization. A second study attempted to determine the subtype selectivity of the M35 antibody by expressing human m1–m5 muscarinic acetylcholine receptors in transfected Chinese hamster ovary cells. The M35 antibody did not show any immunoreactivity in nontransfected control cells (Carsi-Gabrenas et al. 1997). These studies put forth the specificity of the M35 antibody for mAchRs. Cumulatively, it seems the M35-binding epitope is preserved among mAChR subtypes and among species and is likely specific to mAchRs (Carsi-Gabrenas et al. 1997; van der Zee and Luiten 1999). Our Western blot analysis revealed that the M35 antibody also binds to lamprey mAchRs. Using protein extracted from a whole brain of a young adult lamprey, the antibody reacted with bands at 51 and 104 kDa. In the moth, the M35 antibody was shown to react with bands at 85 and 105 kDa (Torkkeli et al. 2005). The 105-kDa band seen in moth and lamprey likely represents a glycosylated form of the receptor (Torkkeli et al. 2005), whereas the 51-kDa band seen in lampreys could arise from partial proteolysis of the mAchR protein because of the extraction method (Venter 1983). We cannot rule out that this band results from binding to a protein other than mAchRs. However, the aforementioned studies have shown that use of the M35 antibody does not reveal nonspecific binding. (Carsi-Gabrenas et al. 1997; Lammerding-Koppel et al. 1995). In mammals, mAchRs were reported with molecular weights between 33 and 86 kDa, depending on the extraction method and glycosylation state of the protein, with 70 kDa being the most frequent observation (Andre et al. 1983, 1984; Avissar et al. 1983; Bartfai et al. 1974; Birdsall et al. 1979; Haga 1980). Our experiments on rat whole brain protein extracts, run in parallel with the lamprey brain protein extracts, served as a positive control. The M35 antibody reacted with a major band at 70 kDa, but also with minor bands between 70 and 86 kDa and between 35 and 51 kDa in rats. Both the lamprey and rat brain tissue showed some M35 binding at 51 kDa. RS neurons within the MRRN or the PRRN reliably oscillated synchronously in response to mAchR activation. This was first suggested by experiments in which bilateral recorded MRRN RS neurons were performed. Calcium imaging of populations of calcium green dextran–filled neurons within the MRRN or PRRN consistently revealed that muscarinic activation of this rhombencephalic network mediates synchronous activity among RS neuron populations. Again, because the RS neurons are not directly activated by muscarine, it suggests that RS neurons are driven by a common source, most likely the mAchR immunoreactive cells discussed above. The synchronous activity within the MRRN or PRRN is clear, but future experiments will need to be carried out to determine whether the MRRN RS neurons oscillate in synchrony with those in the PRRN. Also, it is important to consider that there is some functional heterogeneity within the RS population. It has been shown that different, although overlapping, subpopulations of RS neurons are activated to elicit different motor behaviors (for a review, see Deliagina et al. 2002). As such, future experiments must also be performed to test whether the muscarinoceptive cells drive a subpopulation of or all RS neurons. The most likely hypothesis to explain collectively the results from this study is that muscarine would directly activate muscarinoceptive cells that induce synchronous sustained, recurring depolarizations in bilateral populations of RS neurons. The recurrent aspect of the response is likely caused by application of a high concentration of the exogenous agonists that may not be degraded or may not readily diffuse away from the region of activity. The cellular mechanisms for these sustained depolarizations induced by muscarine have not been worked out in this study. However, Klink and Alonso (1997) described a cell population in layer II of the rat entorhinal cortex in which muscarine directly induces sustained, recurring bursts of activity. Muscarinic receptor activation of the non-stellate, pyramidal-like, cell population of the rat entorhinal cortex induces a slight depolarizing plateau on which individual bursts of 2–5 s recur about every 6 s. Although the period of this activation differs from that of the RS neuron response to muscarine in our study, the burst duration correlates well with our data. In addition to the similar bursting physiology, mAchR activation entrains the cell population within layer II of the rat entorhinal cortex to oscillate synchronously (Dickson and Alonso 1997; Dickson et al. 2000). A similar cell population capable of direct mAchR-induced synchronous, recurring bursting could participate in a lamprey rhombencephalic network that synchronizes the sustained, recurring bursting of the RS neuron population. It was also found in this study that muscarine more reliably induced sustained depolarizations than pilocarpine. This suggests the effect may be preferentially mediated by one mAchR subtype over other subtypes (Kitazawa et al. 1999; McKinney et al. 1991; Schworer et al. 1989). It was proposed that oscillation and synchronization of groups of cells within layer II of the entorhinal cortex could participate in the functional output that is learning and memory (Dickson and Alonso 1997; Dickson et al. 2000). Here we begin to describe a network capable of promoting synchronous depolarizations in a large number of bilateral RS neurons known to activate the spinal locomotor networks in lampreys. A previous study in which RS neuron activity was recorded with chronically implanted electrodes in freely behaving lamprey showed bilateral mass activation occurred irrespective of the modality and laterality of an applied sensory stimulus (Deliagina and Fagerstedt 2000). This bilateral RS neuron mass activation always preceded initiation of locomotion. Similarly, we described a mechanism whereby a unilateral stimulus, here microinjection of muscarine, is capable of inducing bilateral, synchronized activity of the RS system. Also, the muscarine-induced spinal output resembled fictive locomotion. One functional role of the muscarinoceptive cells could be to facilitate transformation of a unilateral sensory input into bilateral RS output. Further experiments are needed to examine the contribution of this muscarine-activated network to lamprey sensorimotor integration. Cholinergic inputs The pattern that is generated under muscarine may not necessarily be a full representation of a particular motor behavior. It is more likely that the circuitry activated by the drug is only part of a network that mediates motor behavior. Locally applied acetylcholine, like muscarine, can induce sustained recurring depolarizations in RS neurons through this network. Under natural conditions, cholinergic neurons located in the lamprey CNS would activate the muscarinoceptive neurons. There are a few potential sources for cholinergic input into the region of muscarinoceptive cells. These inputs include a group of anatomically uncharacterized small, cholinergic cells located near the ventricular surface and medial to the caudal vagal nucleus described by Pombal et al. (2001). Also, according to the latter study, putative preganglionic parasympathetic neurons send cholinergic fibers through this region. The MLR of lampreys is also known to contain numerous cholinergic cells (Le Ray et al. 2003; Pombal et al. 2001). This important region for the control of locomotion was first identified in cats and since then, its presence was shown in many other vertebrate species including lampreys (Bernau et al. 1991; Shik et al. 1966; Sirota et al. 2000; Uematsu and Todo 1997; reviewed in Grillner et al. 1997; Jordan 1998; Shik and Orlovsky 1976). It is a reasonable candidate to provide cholinergic input to the muscarinoceptive cells driving the RS neuron response. The MLR sends descending cholinergic axons into the brain stem reticular formation in mammals (Garcia-Rill and Skinner 1987; Rye et al. 1988). This was shown physiologically in lampreys, where it was shown that MLR stimulation elicits direct nicotinic activation of RS neurons (Le Ray et al. 2003). It was also shown, in the same study that the lamprey MLR contains numerous choline acetyltransferase-immunoreactive cells. Therefore in addition to the direct excitatory drive from the MLR to RS neurons, a parallel activation of the muscarinoceptive cells in the caudal half of the rhombencephalon could enhance the MLR drive onto the RS population and, consequently, increase locomotor drive. Moreover, it has been well documented that a unilateral stimulation of the MLR produces symmetrical locomotion in several species of vertebrates in which this has been examined. The mechanisms by which the transformation of a unilateral input into bilateral activity occurs have not been identified. Therefore the hypothetical bilateral projection from the muscarinoceptive cells to RS neurons could strengthen the bilateral symmetry in the activation of RS neurons by the MLR to produce fluid and symmetrical locomotor output. Experiments are presently under way to test this hypothesis.This study describes components of an important network whereby unilateral mAchR activation of cells in the caudal rhombencephalon induces bilateral synchronous, sustained depolarizations of RS neurons. Glutamatergic transmission is needed for these muscarinoceptive cells to induce this effect, and this may occur between the muscarinoceptive cells and RS neurons. Ongoing efforts to record directly from muscarinoceptive cells in the caudal rhombencephalon should further our understanding of this rhombencephalic network. Also, experiments using a semi-intact preparation could improve our understanding of the contribution of this network to the control of locomotion under more natural conditions. | |||||||||||||||||||
We thank D. Veilleux for assistance with the experiments; F. Bernard for help with the figures; F. Auclair and T. Boutin for the immunohistochemistry experiments; J. Vallée and R. Robitaille for help with the Western blot experiments; and W. D. Swink, M. K. Jones, and R. Bergstedt from the Lake Huron Biological Station, R. McDonald of the Sea Lamprey Control Centre in Sault Ste.Marie, Ontario, and B. Young, E. Howe, and W. Bouffard from the U.S. Fish and Wildlife Service for the supply of lampreys. GRANTS This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-052699 to S. Alford, individual and group grants from the Canadian Institutes of Health Research, and an individual grant from the Natural Sciences and Engineering Research Council of Canada. | |||||||||||||||||||
References
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