AJP - Regu Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 292: R927-R936, 2007. First published November 2, 2006; doi:10.1152/ajpregu.00254.2006
0363-6119/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/2/R927    most recent
00254.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iigaya, K.
Right arrow Articles by Itoh, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iigaya, K.
Right arrow Articles by Itoh, H.

NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Novel axonal projection from the caudal end of the ventrolateral medulla to the intermediolateral cell column

Kamon Iigaya,1 Hiroo Kumagai,1 Hiroshi Onimaru,2 Akira Kawai,2 Naoki Oshima,1 Toshiko Onami,1 Chie Takimoto,1 Tadashi Kamayachi,1 Koichi Hayashi,1 Takao Saruta,1 and Hiroshi Itoh1

1Department of Internal Medicine, Keio University School of Medicine and 2Department of Physiology, Showa University School of Medicine, Tokyo, Japan

Submitted 13 April 2006 ; accepted in final form 30 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We used an optical imaging technique to investigate whether axons of neurons in the caudal end of the ventrolateral medulla (CeVLM), as well as axons of neurons in the rostral ventrolateral medulla (RVLM), project to neurons in the intermediolateral cell column (IML) of the spinal cord. Brain stem-spinal cord preparations from neonatal normotensive Wistar-Kyoto and spontaneously hypertensive rats were stained with a voltage-sensitive dye, and responses to electrical stimulation of the IML at the Th2 level were detected as changes in fluorescence intensity with an optical imaging apparatus (MiCAM-01). The results were as follows: 1) depolarizing responses to IML stimulation during low-Ca high-Mg superfusion were detected on the ventral surface of the medulla at the level of the CeVLM, as well as at the level of the RVLM, 2) depolarizing responses were also detected on cross sections at the level of the CeVLM, and they had a latency of 24.0 ± 5.5 (SD) ms, 3) antidromic action potentials in response to IML stimulation were demonstrated in the CeVLM neurons where optical images were detected, and 4) glutamate application to the CeVLM increased the frequency of excitatory postsynaptic potentials (EPSPs) and induced depolarization of the IML neurons. The optical imaging findings suggested a novel axonal and functional projection from neurons in the CeVLM to the IML. The increase in EPSPs of the IML neurons in response to glutamate application suggests that the CeVLM participates in the regulation of sympathetic nerve activity and blood pressure and may correspond to the caudal pressor area.

optical imaging; patch-clamp technique; monosynaptic bulbospinal projection; caudal pressor area; sympathetic nervous system

BECAUSE NEURONS IN THE intermediolateral cell column (IML) of the spinal cord receive various inputs from higher neurons [the rostral ventrolateral medulla (RVLM), rostral ventromedial medulla (RVMM), paraventricular nucleus, raphe nucleus, etc. (1, 4, 12, 21, 23, 26, 29)], IML neurons play an important role in the regulation of sympathetic outflow. The precise location and function of the caudal pressor area (CPA) have recently been reevaluated, and it is now known to be located more caudally than the caudal ventrolateral medulla (CVLM) (see Refs. 2, 8, 27, 28). Because microinjection of glutamate and homocysteine in the CPA in in vivo experiments induced sympathetic activation and an increase in blood pressure, and microinjection of glycine induced inhibition of sympathetic activity and blood pressure reduction, the neurons in the CPA have been considered to be sympathoexcitatory (16).

Much attention has been focused on afferent inputs in the CPA and efferent pathways out of the CPA. Natarajan and Morrison (16) demonstrated that the CPA neurons project to the sympathoexcitatory CVLM, whose neurons then project to the RVLM. Horiuchi et al. (8) reported that the CPA is modulated by prolactin-releasing peptide, a hypothalamic hormone. Sun and Panneton (27, 28) used anterograde and retrograde tracers to precisely localize the site of the CPA in the ventrolateral medulla (VLM). Li et al. (11) recently found a direct projection from the CPA to the IML in a retrograde tracer experiment. Studies to investigate whether the CPA neurons are anatomically and functionally bulbospinal neurons have just begun.

In the present study, we used optical imaging, electrophysiological, and histological methods to determine whether an axonal projection exists from the medulla oblongata, including the CPA, to the IML. Because we did not measure blood pressure in this in vitro study, we will refer to the area as the caudal end of the VLM (CeVLM), instead of as the CPA. Using optical imaging, we tried to detect depolarizing responses on the ventral surface of the medulla oblongata and cross sections of brain stem-spinal cord preparations to electrical stimulation of the axons or neural terminals of presympathetic neurons that projected to the IML region (IML stimulation). We also examined antidromic action potentials in response to the IML stimulation in the CeVLM area, in which optical imaging showed depolarizing responses. The preliminary results have been presented in the form of an abstract (9).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparations and staining. Experiments were performed on brain stem-spinal cord preparations from 2- to 4-day-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) (see Refs. 14, 15, and 21). The experimental protocols were approved by the Animal Research Committee of Keio University School of Medicine in compliance with Japanese Law (no. 105). Under deep ether anesthesia, the brain stem and spinal cord were isolated as previously described (14), and the spinal cord was sectioned at the second thoracic nerve root (Th2) level (Fig. 1). The following three different types of preparations were used: 1) a type in which the brain stem was sectioned at the distal end of the pons for observation of the ventral surface of the medulla oblongata, 2) a type in which the brain stem was sectioned between the posterior inferior cerebellar artery and the top of the hypoglossal nerve roots for observation of cross sections at the RVLM level, and 3) a type in which the brain stem was sectioned between the branch point of the basilar artery and the distal end of the exit points of the hypoglossal nerve roots for observation of cross sections at the level of the CeVLM.


Figure 1
View larger version (45K):
[in this window]
[in a new window]

  		Fig. 1. Brain stem-spinal cord preparation from a neonatal spontaneously hypertensive rat. Three different types of preparations were used for optical imaging and the whole cell patch-clamp technique as follows: 1) a type for observation on the ventral surface of the medulla oblongata, 2) a type for observation of cross sections at the level of the rostral ventrolateral medulla (RVLM), and 3) a type for observation of cross sections at the level of the caudal end of the VLM (CeVLM). Electrical stimulation was applied to the intermediolateral cell column (IML) at the Th2 level.

 
The preparations were continuously superfused at 2–3 ml/min with a standard solution, pH 7.4, consisting of (in mmol/l) 124 NaCl, 5.0 KCl, 2.4 CaCl2, 1.3 MgCl2, 26 NaHCO3, 1.2 KH2PO4, and 30 D-glucose and equilibrated with 95% O2 and 5% CO2 at 26–27°C. In most of the experiments, the preparations were superfused with a low-Ca high-Mg solution containing (in mmol/l) 0.2 CaCl2 and 5.0 MgCl2 to exclude ascending monosynaptic and polysynaptic projections and descending polysynaptic projections and to detect descending monosynaptic projections alone.

Optical imaging. The brain stem-spinal cord preparations were incubated for 40–50 min in the standard solution containing the fluorescent voltage-sensitive dye Di-4-ANEPPS (0.1 mg/ml) or Di-2-ANEPEQ (50 µg/ml; Molecular Probes, Eugene, OR; see Refs. 19, 20, 30–32). The hydrophilic dye Di-2-ANEPEQ was used for observation at the brain stem surface because of its higher diffusion property into tissue than the lipophilic dye Di-4-ANEPPS. Di-4-ANEPPS was used for observation at the cut surface of the transverse sections because it provides more stable staining with less noise than Di-2-ANEPEQ. Previous study reported that the depth for optical detection of spontaneous bursting activity was <500 µm and that the effective depth might be 200–300 µm for Di-2-ANEPEQ (20). The depth for optical detection of responses to electrical stimulation may be greater than for spontaneous burst activity.

The preparation was then placed in a perfusion chamber (volume 1 ml) mounted on a fluorescence microscope (BX50WIF-2; Olympus Optical, Tokyo, Japan), with the ventral surface or cross section of the medulla and spinal cord facing up.

Neuronal activity in the preparation was detected as a change in fluorescence of the voltage-sensitive dye with an optical imaging apparatus (MiCAM01; Brain Vision, Tsukuba, Japan; see Refs. 19, 20, 31, 32) and a tungsten-halogen lamp (150 watts) through a 510–550-nm excitation filter, dichroic mirror, and 590-nm absorption filter (U-MWIG2 mirror unit; Olympus Optical). The head of charge-coupled device (CCD) camera had an 8.4 x 6.5 mm2 imaging area consisting of 180 x 120 pixels and a maximal time resolution of 3.5 ms. The power of the microscope was adjusted to x2 in most experiments, so that the image sensor could cover an area measuring 4.2 x 3.25 mm2. We examined optical images of the medulla oblongata for responses to electrical stimulation of the IML. Very thin stainless steel electrodes (tip diameter 20 µm, length 100 µm, impedance 700 k{Omega}) were used, and the position of the IML neurons was carefully identified through the CCD camera to enable stimulation (10–50 volts, 100 µs, single pulse) of only axons that projected to the IML neurons as much as possible by inserting the electrode perpendicularly in cross sections of the spinal cord (Fig. 1). The IML was stimulated at the Th2 level, which is known to be the main target region of the cardiac sympathetic premotor neurons in the brain stem.

Most recordings were performed with an acquisition time of 10 ms. Fluorescence signals during a 3.4-s period/trial were totaled and averaged for 10–20 electric stimulations of ipsilateral IML neurons. The fluorescence changes are expressed as ratios (fluorescence intensity divided by the fluorescence intensity of the reference image). The differential images were processed with a software-spatial filter for 2 x 2 pixels and presented as pseudocolor displays in which the "red" region corresponded to a decrease in fluorescence, meaning membrane depolarization. The intensity of the depolarizing response was strongest in the red region, grew weaker in the yellow region, and still weaker in the green region. Decreases in fluorescence (depolarization) are presented as upward of the intensity of the depolarization. Because optical signals basically represent depolarization of the soma and not of the axons (30), we refer to the fluorescence change as "a depolarizing response." Values are reported as means ± SD. Data were compared with assessment of statistical significance by the unpaired t-test.

Electrophysiological experiments. We investigated whether the neurons in the CeVLM that exhibited depolarizing responses to IML stimulation detected by optical imaging projected to the IML. Neuronal activity was recorded extracellularly or intracellularly during low-Ca high-Mg superfusion (21), and the IML was stimulated to investigate the induction of antidromic action potentials. For the extracellular recordings, glass electrodes were filled with 2% pontamine sky blue in 0.5 mol/l sodium acetate (resistance 5–20 M{Omega}). For the intracellular recordings by the whole cell patch-clamp technique, patch electrodes were filled with 0.5% lucifer yellow in a pipette solution of the following composition (mmol/l): 130 potassium gluconate, 10 EGTA, 10 HEPES, 2 Na2-ATP, 1 CaCl2, and 1 MgCl2, with pH 7.2–7.3 adjusted with KOH. The IML was stimulated with a stainless steel electrode (5–15 volts, 100 µs, single pulse) to induce antidromic action potentials in the VLM.

In addition, to examine functional and orthodromic effects of CeVLM neurons on the IML, IML neurons were recorded by an intracellular technique (whole cell patch-clamp) and an extracellular technique during superfusion with standard solution. Glutamate (1 mmol/l, 10–30 µl) was locally applied to the CeVLM in a preparation whose cross section was at the level of the CeVLM and from which more rostral regions, including the CVLM and RVLM, have been removed (type 3 preparation under Preparations and staining and Fig. 1). To confirm and minimize glutamate diffusion in the cross section at the level of the CeVLM, the glutamate solution was mixed with Fast Blue.

Histological examination. After immersing the preparations in 10% formalin-phosphate buffer solution at 4°C for 48 h, the medulla oblongata with spinal cord was cut into 100-µm sections, and the locations of neurons that had stained with lucifer yellow or pontamine sky blue were identified in the medulla and spinal cord. All preparations were then stained with neutral red to compare the location of the CeVLM neurons with the location of surrounding nuclei.

At the conclusion of the optical imaging experiments, the IML that had been electrically stimulated was coagulated (20 volts, 20 ms, single pulse) to confirm that the neurons that projected to the IML had been accurately stimulated.

To confirm that the neurons in the spinal cord that depolarized in response to glutamate application to the CeVLM were IML neurons, they were stained with lucifer yellow at the conclusion of the intracellular recording.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Optical imaging on the ventral surface. Depolarizing responses were actually captured in the form of a motion picture. As shown in Fig. 2, during superfusion with standard solution, IML stimulation induced depolarizing responses on the continuous column of the surface of the rostrocaudal VLM, including the RVLM, the CVLM, and the CeVLM. In all preparations (n = 10), the intensity of depolarization (fluorescence change) of the CeVLM (–0.098 ± 0.024%, means ± SD) was significantly (P < 0.01) stronger than that in the RVLM (–0.048 ± 0.024%) and CVLM. The latency between IML stimulation and the start and the peak of the depolarizing response at the CeVLM was 41 ± 15 and 82 ± 32 ms, respectively. No depolarizing responses were detected in other regions despite even stronger stimulation of the IML.


Figure 2
View larger version (63K):
[in this window]
[in a new window]

  		Fig. 2. Optical images of the ventral surface of the medulla oblongata during superfusion with standard solution. Depolarizing responses to IML stimulation were clearly detected on the ventral surface at the RVLM, the caudal ventrolateral medulla (CVLM), and the CeVLM (n = 10).

 
As shown in Fig. 3, the IML stimulation during low-Ca high-Mg superfusion also induced depolarizing responses on the surface of the rostrocaudal VLM, including the RVLM, the CVLM, and the CeVLM. The low-Ca high-Mg solution was used to exclude ascending monosynaptic and polysynaptic projections and descending polysynaptic projections and obtain only the depolarizing responses of descending monosynaptic projections. In all preparations (n = 6), the depolarizing response of the CeVLM (–0.041 ± 0.009%) was significantly (P < 0.05) stronger than that of the RVLM (–0.026 ± 0.010%). The latency between IML stimulation and the start and the peak of depolarization in the CeVLM was 28 ± 7 and 31 ± 11 ms, respectively. The intensity of the depolarizing response was weaker during low-Ca high-Mg superfusion than during perfusion with the standard solution.


Figure 3
View larger version (67K):
[in this window]
[in a new window]

  		Fig. 3. Optical images of the ventral surface of the medulla oblongata during superfusion with low-Ca high-Mg solution. Depolarizing responses to IML stimulation were detected on the ventral surface at the RVLM, the CVLM, and the CeVLM (n = 6).

 
Optical imaging of the cross sections. IML stimulation during low-Ca high-Mg superfusion induced depolarizing responses in restricted regions on cross sections at the level of the RVLM (n = 6; Fig. 4) and at the level of the CeVLM (n = 8; Fig. 5). The latency between IML stimulation and the start and the peak of the depolarizing response was 24 ± 5 and 36 ± 8 ms, respectively, at the level of the CeVLM. That was 27 ± 9 and 35 ± 10 ms, respectively, at the level of the RVLM.


Figure 4
View larger version (66K):
[in this window]
[in a new window]

  		Fig. 4. Optical images of a cross section at the level of the RVLM during low-Ca high-Mg superfusion. A depolarizing response to IML stimulation was detected at the RVLM (n = 6). The latency between stimulation of the IML and the start and the peak of the depolarizing response at the RVLM was 27 ± 9 and 35 ± 10 (SD) ms, respectively.

 

Figure 5
View larger version (62K):
[in this window]
[in a new window]

  		Fig. 5. Optical images of a cross section at the level of the CeVLM during low-Ca high-Mg superfusion. A depolarizing response to IML stimulation was detected at the CeVLM (n = 8). The latency (means ± SD) between stimulation of the IML and the start and the peak of depolarizing response at the CeVLM was 24 ± 5 and 36 ± 8 ms, respectively.

 
To determine whether the depolarizing responses were derived from the somas and not from the axons, we examined the depolarizing responses after adding {gamma}-aminobutyric acid (GABA) to the superfusion solution. IML stimulation during superfusion with low-Ca high-Mg solution containing GABA (200 µmol/l, n = 7) did not induce any depolarizing responses in the cross sections at the level of the CeVLM (Fig. 6) or of the RVLM.


Figure 6
View larger version (87K):
[in this window]
[in a new window]

  		Fig. 6. Optical images of a cross section at the level of the CeVLM before (top) and during {gamma}-aminobutyric acid (GABA) superfusion (bottom). IML stimulation during superfusion with low-Ca high-Mg solution containing GABA (200 µmol/l, n = 7) did not induce any depolarizing responses at the CeVLM.

 
Electrophysiological experiments. Other brain stem-spinal cord preparations were fashioned for the electrophysiological experiments, and during low-Ca high-Mg superfusion antidromic action potentials in response to IML stimulation were clearly detected in 9 CeVLM neurons by intracellular recordings (whole cell patch-clamp technique; Fig. 7) and in 16 CeVLM neurons by extracellular recordings. The latency between IML stimulation and detection of the antidromic action potential was 21.6 ± 7.1 ms by extracellular recording and 27.2 ± 4.6 ms by the whole cell patch-clamp technique. These latencies are almost the same as the latency between IML stimulation and the depolarizing response in the CeVLM measured by optical imaging of cross sections (see Fig. 5). The spontaneous action potentials of the CeVLM neurons (extracellular recording) collided with subsequent antidromically evoked action potentials.


Figure 7
View larger version (5K):
[in this window]
[in a new window]

  		Fig. 7. Antidromic action potential of a neuron in the CeVLM recorded by the whole cell patch-clamp technique in response to electrical stimulation of the IML at the Th2 level. The latency between stimulation and the start of the antidromic action potential was 27.2 ± 4.6 ms according to the whole cell patch-clamp recordings (n = 9).

 
Glutamate application to the CeVLM. During whole cell patch-clamp recordings of neurons in the IML with standard solution (Fig. 8A), glutamate was applied to the CeVLM locally after removing more rostral regions, including the CVLM and RVLM (type 3 preparation, as in Figs. 5 and 6). Local application of glutamate to the CeVLM increased the frequency of the excitatory postsynaptic potentials (EPSPs) and induced significant depolarization (5.6 ± 2.5 mV) of the IML neurons (n = 8; Fig. 8A). Each EPSP was clearly revealed by increasing the speed of the sweep (Fig. 8C).


Figure 8
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 8. Intracellular recordings of a neuron in the IML at the Th2 level (n = 8) during superfusion with standard solution, in the type 3 preparation, like in Figs. 5 and 6. A: local application of glutamate (1 mmol/l) to the CeVLM increased the number of excitatory postsynaptic potentials (EPSPs), induced depolarization (5.63 ± 2.50 mV), and increased the action potential spikes of the IML neurons. B: few EPSPs were seen before glutamate application. C: each EPSP and depolarization was clearly detected after local application of glutamate to the CeVLM.

 
Histological examination. The locations of the neurons in the CeVLM in which depolarizing responses were detected by optical imaging and that exhibited an antidromic action potential in response to IML stimulation were investigated histologically (Fig. 9A). The neurons stained with lucifer yellow or pontamine sky blue were located in the lateral side of the lateral reticular nucleus (LRt) or in the LRt, the caudal part of the nucleus ambiguus, and the medial side of trigeminal spinal tract nucleus at the level of pyramidal decussation (Fig. 9, B and C). Thus the anatomic location of the neurons in the CeVLM satisfied the criteria for the location of the CPA described by Sun and Panneton (27, 28).


Figure 9
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 9. A: location of neurons in the CeVLM that exhibited an antidromic action potential in response to electrical stimulation of the IML. This experiment was carried out during low-Ca high-Mg superfusion. bullet, Extracellular recordings; {circ}, intracellular recordings. AP, area postrema; 12, hypoglossal nucleus; Sp5C, spinal trigeminal nucleus, caudal part; pyx, pyramidal decussation; LRt, lateral reticular nucleus; RAmb, retroambiguus nucleus; IO, inferior olivary nucleus; IOM, inferior olive, medial nucleus. B: fluorescence image showing a neuron in the CeVLM that exhibited an antidromic action potential and stained with lucifer-yellow. C: drawing of the location of a neuron that exhibited an antidromic action potential and that was stained with lucifer yellow.

 
We tried to confirm that the location of the electrical stimulation in the spinal cord actually corresponded to the IML, and the results showed that location of the electrical stimulation and coagulation did correspond to the location of IML neurons (Fig. 10, A and B).


Figure 10
View larger version (106K):
[in this window]
[in a new window]

  		Fig. 10. A: histological examination of the location of IML neurons at the Th2 level that were coagulated at the conclusion of electrical stimulation of the IML. Shown is a cross section stained with neutral red. Top: dorsal; bottom, ventral. B: high-magnification image of A showing the location of electrical stimulation of the IML. The location that was electrically stimulated and then coagulated (indicated by the arrow) actually corresponded to the IML neurons. C: a neuron in the IML that exhibited depolarization and an increase in EPSPs in response to glutamate application to the CeVLM. This neuron stained with lucifer yellow is the same neuron shown in Fig. 8. D: cross section at the Th2 level stained with neutral red. The neuron that exhibited the increase in EPSPs, stained with lucifer yellow, and shown in C was actually located in the IML region ({circ}). Top: dorsal; bottom, ventral.

 
The location of neurons that exhibited depolarization and an increase in EPSP in response to local glutamate application to the CeVLM and stained with lucifer yellow in the spinal cord also corresponded to the IML (Fig. 10, C and D).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Characteristics of neurons in the CeVLM identified by optical imaging. We used optical imaging and electrophysiological methods in brain stem-spinal cord preparations of neonatal SHRs to determine whether neurons in the CeVLM axonally project to the IML. The optical imaging revealed strong depolarizing responses in the CeVLM to IML stimulation, and CeVLM neurons fired antidromic action potentials in response to the IML stimulation. Glutamate applied in the CeVLM induced depolarization in the IML neurons. These findings demonstrated a monosynaptic axonal and excitatory projection from the CeVLM to neurons in the IML.

A number of studies, including a retrograde tracer study (10), have reported a monosynaptic projection from the RVLM, the RVMM, and the raphe nucleus to the IML (1, 4, 23, 26). Several studies have also demonstrated a direct projection from the CVLM to the IML in adult rats (7, 13), and the result of a recent tracer study using cholera toxin B subunit suggested a projection from the CPA to the IML of adult rats (11). A new region that differs from the CPA, the medullocervical pressor area, has recently been reported to project to the IML in adult rats based on an in vivo retrograde tracer study (25). Consistent with these findings, the optical imaging in our study suggested that a continuous longitudinal rostrocaudal column in the VLM, including the RVLM, the CVLM, and the CeVLM, gives rise to a monosynaptic projection to the IML. Because we detected the strongest depolarizing response in the CeVLM, we focused specifically on the CeVLM in the present study. To our knowledge, few studies have succeeded in optical visualization of the axonal projection from the CeVLM neurons that are involved in sympathetic nerve regulation to the IML. Because the existence of the projection from the CPA to the IML had been suggested only by a tracer study (11), the projection we have found must be discussed critically.

Earlier studies have shown that EPSPs or postsynaptic responses completely disappeared during superfusion with low-Ca high-Mg solution, suggesting a complete blockade of synaptic transmission (6, 17, 22). In our unpublished observations, many EPSPs were detected in the CeVLM neuron in response to IML stimulation during superfusion with standard solution, whereas, during superfusion with low-Ca, high-Mg solution, the EPSPs observed in the CeVLM neuron to IML stimulation completely disappeared. Thus use of a low-Ca high-Mg superfusion has the advantage of blocking all synapses and enabling detection of only descending monosynaptic projections alone, whereas, during superfusion with standard solution, depolarizing responses of descending polysynaptic projections and ascending projections are detected as well as descending monosynaptic projections. In the present study, the regions that exhibited depolarizing responses during low-Ca high-Mg superfusion were the same as those that displayed them during superfusion with the standard solution. In contrast, the depolarizing responses were less intense during low-Ca high-Mg superfusion, and the peak time of the depolarizing responses during low-Ca high-Mg superfusion occurred earlier than with the standard solution. These results strongly suggest that neurons in the CeVLM monosynaptically project to the IML neurons.

Previous studies found that low-Ca high-Mg solution did not affect neuronal excitability (6, 18). If neuronal excitability decreases during superfusion with low-Ca high-Mg solution, the intensity of the depolarizing responses detected in the present study should be weaker than the actual depolarizing responses. Therefore, we did not overestimate the depolarizing response by optical imaging.

Optical imaging findings on the ventral surface revealed that the depolarizing response in the CeVLM was more intense than in the RVLM. This finding suggests that, in neonatal rats, more neurons project from the CeVLM to the IML than from the RVLM and CVLM to the IML but does not imply that neuronal activity in the CeVLM is stronger than in the RVLM. It may merely reflect the characteristics of neonatal rats. However, we were unable to compare the depolarizing responses during the different developmental stages of the rats in this study.

Because the depolarizing response is the sum of the signals in the somas (cell bodies) and axon bundles, we investigated whether the depolarizing response originated in the somas or in the axons. GABA should bind to receptors on the surface of the soma, and, as shown in Fig. 6, the depolarizing response of the CeVLM was completely abolished during superfusion with GABA. This finding suggests that the depolarizing responses detected by optical imaging reflected depolarization of the somas of CeVLM neurons, not depolarization of the axons. Because we have reported the existence of the CeVLM neurons for the first time, it is unknown whether GABA receptors are present on CeVLM neurons. However, the result that the IML stimulation during superfusion with low-Ca high-Mg solution containing GABA did not induce any depolarizing responses in the cross sections of the CeVLM suggests to us that the CeVLM neurons possess GABA receptors. An earlier study reported that CPA neurons possess GABA receptors (24), and, if the CeVLM neurons correspond to CPA neurons, then CeVLM neurons probably possess GABA receptors.

We also obtained some optical imaging data in WKY rats as a control for the SHRs. The data showed similar depolarizing responses to IML stimulation in the CeVLM on the ventral surface (n = 5) and cross sections (n = 4) of WKY rats. Although careful experiments are required to make quantitative comparisons between the intensity of depolarizing responses in the CeVLM of WKY rats and SHRs, the results indicate to us that the projection from the CeVLM to the IML exists in both normotensive and hypertensive neonatal rats. It would be interesting to compare responses in the IML neurons of WKY rats and SHRs when glutamate is locally applied to the CeVLM on the preparation in the absence of the CVLM and the RVLM regions.

Methodological limitations. We cannot completely rule out the possibility that we may have stimulated regions other than the axons and neural terminals of bulbospinal neurons in the IML region (e.g., ascending neurons). However, for the following reasons, we think that the extent of the electrical stimulation in this study was relatively restricted to IML. We used very thin electrodes and carefully identified and stimulated the position of the IML neurons through the CCD camera so as to limit the stimulation point. Indeed, as shown in Fig. 10, we demonstrated that the stimulation point was accurately restricted to within the IML region. The location of the depolarizing response in Fig. 4 therefore includes RVLM neurons. The depolarizing response that was detected in the region of the RVLM in response to IML stimulation served as a good positive control.

These results also suggested that the stimulation point accurately covered axons and neural terminals of bulbospinal neurons in the IML. We were therefore able to conclude that the depolarizing responses detected in the CeVLM on both the ventral surface and cross section were depolarizations of bulbospinal neurons. As shown in Fig. 8, we also demonstrated EPSPs on the IML neurons detected during chemical stimulation of the CeVLM. The results confirmed the occurrence of orthodromic responses, demonstrating the presence of a functional projection from the CeVLM to the IML.

Developmental issues. Although a tracer study (10) found that neurons in the RVMM and in the raphe nucleus project to the IML, we did not detect any depolarizing responses in the RVMM or the raphe nucleus after IML stimulation. Because the intensity of the depolarizing response depends on the number of neurons that respond, the number of neurons projecting from these areas to the IML may be relatively small. For example, in the raphe nucleus of rabbits between day 26 of gestation and 6 days of age, the dendrites showed expansion, increased the number and length of neurons, and developed abundant spines (5). During this period, the soma grew in size. After postnatal day 6 to adulthood, a mature pattern of dendritic branching was achieved. Therefore, the reason that we were unable to detect depolarizing responses in the midline raphe may be because of the immaturity of the neonatal rats.

Second, a previous study showed that gap junctions between neurons may be more abundant in newborn rats than in adult rats (3), and the depolarizing responses may have been exaggerated by the gap junctions because we used newborn rats that were only 2–4 days old. The decrease in gap junctions during development may weaken the depolarizing response.

Physiological implications. In this in vitro study, we investigated whether the CeVLM corresponds to the CPA, an area that has been identified in in vivo studies (2, 8, 16, 27, 28). However, few in vitro studies have identified the anatomic location of the CPA. Our histological examination demonstrated that the location of the CeVLM neurons that exhibited an antidromic action potential and stained with lucifer yellow or pontamine sky blue corresponded to the location where depolarizing responses were observed by optical imaging. The histological examination also showed that the location of the depolarizing responses in the CeVLM neurons was almost identical to that of the CPA reported in earlier in vivo studies (11, 27, 28).

To further confirm that the CeVLM corresponds to the CPA, we investigated whether neurons in the CeVLM are involved in peripheral sympathetic nerve regulation. The IML neurons exhibited increased EPSPs and membrane depolarization in response to local application of glutamate to the CeVLM despite the absence of the RVLM and the CVLM. These results imply that neurons in the CeVLM send excitatory input to the IML neurons and that CeVLM neurons are involved in sympathetic nerve regulation and blood pressure control. If these implications are true, we can conclude that at least some of the neurons in the CeVLM correspond to neurons in the CPA. Further study by spike-triggered averaging is needed to determine whether the spontaneously occurring action potentials of the CeVLM neurons induce EPSPs in the IML neurons.

Because earlier studies have demonstrated that the functions of the CPA are mediated via sympathoinhibitory (2) and sympathoexcitatory (16) neurons in the CVLM, the CPA neurons would be expected to project to the CVLM. However, if the CeVLM corresponds to the CPA, our optical imaging findings suggest that the CPA also projects to the IML.

Even if the CeVLM does not correspond to the CPA, the CeVLM may play a role in sympathetic nerve modulation. It is hard to imagine that the CeVLM affects sympathetic nerve activity in the same manner as the neurons in the RVLM do, and instead we postulate that the CeVLM neurons modulate sympathetic nerve activity determined by the RVLM. Horiuchi et al. (8) found that microinjection of prolactin-releasing peptide in the CPA increased blood pressure but that microinjection of ANG II did not. Thus some functions of the CeVLM, such as responses to stress, differ from those of the RVLM neurons.

In summary, the optical imaging findings in the brain stem-spinal cord preparations of neonatal SHRs suggest the existence of a continuous longitudinal column in the VLM, including the RVLM, the CVLM, and the CeVLM, that gives rise to a monosynaptic projection to the IML. The neurons in the CeVLM have a role in sympathetic nerve and blood pressure control. The projection pathway and function of the CeVLM neurons need to be investigated more precisely in a future study.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from the Kimura Memorial Heart Foundation for research on the autonomic nervous system and hypertension.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Kumagai, Dept. of Internal Medicine, Keio Univ. School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan (e-mail: hkumagai{at}sc.itc.keio.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Cabot J. Sympathetic preganglionic neurons: cytoarchitecture, ultrastructure and biophysical properties. In: Central Regulation of Autonomic Functions, edited by Loewy AD and Spyer KM. New York: Oxford Univ Press, 1990, p. 44–68.
  2. Campos RR Jr, Possas OS, Cravo SL, Lopes OU, Guertzenstein PG. Putative pathways involved in cardiovascular responses evoked from the caudal pressor area. Braz J Med Biol Res 27: 2467–2479, 1994.[ISI][Medline]
  3. Chang Q, Gonzalez M, Pinter MJ, Balice-Gordon RJ. Gap junctional coupling and patterns of connexin expression among neonatal rat lumbar spinal motor neurons. J Neurosc 19: 10813–10828, 1999.[Abstract/Free Full Text]
  4. Dembowsky K, Czachurski J, Seller H. An intracellular study of the synaptic input to sympathetic preganglionic neurones of the third thoracic segment of the cat. J Appl Neurosci 13: 201–244, 1985.
  5. Felten DL, Cummings JP. Ontogeny of medullary raphe nuclei in the rabbit brain stem: a Golgi study. Brain Res Bull 6: 413–425, 1981.[CrossRef][ISI][Medline]
  6. Haas HL, Jefferys JG. Low-calcium field burst discharges of CA1 pyramidal neurons in rat hippocampal slices. J Physiol 354: 185–201, 1984.[Abstract/Free Full Text]
  7. Hardy SG, Horecky JG, Presley KG. Projections of the caudal ventrolateral medulla to the thoracic spinal cord in the rat. Anat Rec 250: 95–102, 1998.[CrossRef][Medline]
  8. Horiuchi J, Saigusa T, Sugiyama N, Kanba S, Nishida Y, Sato Y, Hinuma S, Arita J. Effects of prolactin-releasing peptide microinjection into the ventrolateral medulla on arterial pressure and sympathetic activity in rats. Brain Res 958: 201–209, 2002.[CrossRef][ISI][Medline]
  9. Iigaya K, Kumagai H, Onimaru H, Kawai A, Oshima N, Onami T, Takimoto C, Saruta T. Optical imaging technique revealed a novel axonal projection from caudal end of ventrolateral medulla (VLM) to intermediolateral cell column (IML) in the sympathetic regulation (Abstract). Hypertension 46: 831, 2005.
  10. Jansen AS, Wessendorf MW, Loewy AD. Transneuronal labeling of CNS neuropeptide and monoamine neurons after pseudorabies virus injections into the stellate ganglion. Brain Res 683: 1–24, 1995.[CrossRef][ISI][Medline]
  11. Li Q, Goodchild AK, Seyedabadi M, Pilowsky PM. Preprotachykinin A mRNA is colocalized with tyrosine hydroxylase-immunoreactivity in bulbospinal neurons. Neuroscience 136: 205–216, 2005.[CrossRef][ISI][Medline]
  12. Lipski J, Kawai Y, Qi J, Comer A, Win J. Whole cell patch-clamp study of putative vasomotor neurons isolated from the rostral ventrolateral medulla. Am J Physiol Regul Integr Comp Physiol 274: R1099–R1110, 1998.[Abstract/Free Full Text]
  13. Matsumoto M, Takayama K, Miura M. Distribution of glutamate- and GABA-immunoreactive neurons projecting to the vasomotor center of the intermediolateral nucleus of the lower thoracic cord of Wistar rats: a double-labeling study. Neurosci Lett 174: 165–816, 1994.[CrossRef][ISI][Medline]
  14. Matsuura T, Kumagai H, Kawai A, Onimaru H, Imai M, Oshima N, Sakata K, Saruta T. Rostral ventrolateral medulla neurons of neonatal Wistar-Kyoto and spontaneously hypertensive rats. Hypertension 40: 560–565, 2002.[Abstract/Free Full Text]
  15. Matsuura T, Kumagai H, Onimaru H, Kawai A, Iigaya K, Onami T, Sakata K, Oshima N, Sugaya T, Saruta T. Electrophysiological properties of rostral ventrolateral medulla neurons in angiotensin II 1a receptor knockout mice. Hypertension 46: 349–354, 2005.[Abstract/Free Full Text]
  16. Natarajan M, Morrison SF. Sympathoexcitatory CVLM neurons mediate responses to caudal pressor area stimulation. Am J Physiol Regul Integr Comp Physiol 279: R364–R374, 2000.[Abstract/Free Full Text]
  17. Onimaru H, Arata A, Homma I. Firing properties of respiratory rhythm generating neurons in the absence of synaptic transmission in rat medulla in vitro. Exp Brain Res 76: 530–536, 1989.[CrossRef][ISI][Medline]
  18. Onimaru H, Arata A, Homma I. Intrinsic burst generation of preinspiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats. Exp Brain Res 106: 57–68, 1995.[ISI][Medline]
  19. Onimaru H, Homma I. A novel functional neuron group for respiratory rhythm generation in the ventral medulla. J Neurosci 23: 1478–1486, 2003.[Abstract/Free Full Text]
  20. Onimaru H, Homma I. Optical imaging of respiratory neuron activity from the dorsal view of the lower brainstem. Clin Exp Pharmacol Physiol 32: 297–301, 2005.[CrossRef][ISI][Medline]
  21. Oshima N, Kumagai H, Kawai A, Sakata K, Matsuura T, Saruta T. Three types of putative presympathetic neurons in the rostral ventrolateral medulla studied with rat brainstem-spinal cord preparation. Auton Neurosci 84: 40–49, 2000.[CrossRef][ISI][Medline]
  22. Otsuka M, Yanagisawa M. The effects of substance P and baclofen on motoneurones of isolated spinal cord of the newborn rat. J Exp Biol 89:201–214, 1980.[Abstract/Free Full Text]
  23. Pilowsky P, Llewellyn-Smith IJ, Arnolda L, Inson J, Chalmers J. Intracellular recording from sympathetic preganglionic neurons in cat lumbar spinal cord. Brain Res 656: 319–328, 1994.[CrossRef][ISI][Medline]
  24. Possas OS, Guertzenstein PG. A fall in arterial blood pressure produced by inhibition of the caudal most ventrolateral medulla: the caudal pressor area. J Auton Nerv Syst 49: 235–234, 1994.[CrossRef][ISI][Medline]
  25. Seyedabadi M, Li Q, Padley JR, Pilowsky PM, Goodchild AK. A novel pressor area at the medullo-cervical junction that is not dependent on the RVLM: efferent pathways and chemical mediators. J Neurosci 26: 5420–5427, 2006.[Abstract/Free Full Text]
  26. Strack AM, Sawyer WB, Hughes JH, Platt KB, Loewy AD. A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections. Brain Res 491: 156–162, 1989.[CrossRef][ISI][Medline]
  27. Sun W, Panneton WM. The caudal pressor area of the rat: its precise location and projections to the ventrolateral medulla. Am J Physiol Regul Integr Comp Physiol 283: R768–R778, 2002.[Abstract/Free Full Text]
  28. Sun W, Panneton WM. Defining projections from the caudal pressor area of the caudal ventrolateral medulla. J Comp Neurol 482: 273–293, 2005.[CrossRef][ISI][Medline]
  29. Sved AF, Ito S, Madden CJ, Stocker SD, Yajima Y. Excitatory inputs to the RVLM in the context of the baroreceptor reflex. Ann NY Acad Sci 940: 247–258, 2001.[Abstract/Free Full Text]
  30. Tokumasu M, Nakazono Y, Ide H, Akagawa K, Onimaru H. Optical recording of spontaneous respiratory neuron activity in the rat brain stem. Jpn J Physiol 51: 613–619, 2001.[CrossRef][ISI][Medline]
  31. Tominaga T, Tominaga Y, Ichikawa M. Simultaneous multi-site recordings of neural activity with an inline multi-electrode array and optical measurement in rat hippocampal slices. Pflugers Arch 443: 317–322, 2001.[CrossRef][ISI][Medline]
  32. Tominaga T, Tominaga Y, Ichikawa M. Optical imaging of long-lasting depolarization on burst stimulation in area CA1 of rat hippocampal slices. J Neurophysiol 88: 1523–1532, 2002.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/2/R927    most recent
00254.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iigaya, K.
Right arrow Articles by Itoh, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iigaya, K.
Right arrow Articles by Itoh, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.