Activation of brainstem catecholaminergic neurons during voluntary diving in rats

Brain Research 984 (2003) 42–53 www.elsevier.com / locate / brainres

Research report

Activation of brainstem catecholaminergic neurons during voluntary diving in rats Paul F. McCulloch a , *, W. Michael Panneton b a

Department of Physiology, Midwestern University, 555 31 st Street, Downers Grove, IL 60515, USA b Department of Anatomy and Neurobiology, Saint Louis University, St. Louis, MO 63104, USA Accepted 22 May 2003

Abstract Underwater submergence produces a complex autonomic response that includes apnea, a parasympathetically-mediated bradycardia, and a sympathetically-mediated increase in total peripheral resistance (TPR). The present study was designed to identify brainstem catecholaminergic neurons that may be involved in producing the increased TPR during underwater submergence. Twelve male Sprague–Dawley rats were trained to voluntarily dive 5 m through an underwater maze. On the day of the experiment the rats were randomly separated into a Diving group that repetitively dived underwater, a Swimming group that repetitively swam on the surface of the water, and a Control group that remained in their cages. After the experiment the brainstems of the rats were immunohistologically processed for Fos as an indicator of neuronal activation, and for tyrosine hydroxylase (TH) as an indentifier of catecholaminergic neurons. Neurons labeled with both Fos and TH identified activated catecholaminergic neurons. In Diving rats there was increased Fos1TH labeling in A1, C1, A2, A5, and sub-coeruleus, as well as globosa neurons in the lateral A7 region compared with Control rats, and in A1, C1 and A5 compared with Swimming rats. In Swimming rats Fos1TH labeling was significantly increased in caudal A1, A5, sub-coeruleus and globosa neurons compared with Control rats. These data suggest that selective groups of catecholaminergic neurons within the brainstem are activated by voluntary underwater submergence, and some probably contribute to the sympathetically-mediated increase in vascular tone during diving.  2003 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation; Respiratory regulation Keywords: Catecholamine; Tyrosine hydroxylase; Fos; Autonomic nervous system; Diving response; Rat

1. Introduction Underwater submergence produces a complex of autonomic reflexes, the ‘diving response’, which includes an apnea, a parasympathetically-mediated bradycardia, and a sympathetically mediated increase in peripheral vascular tone. During underwater submergence, cardiac output is generally reduced in proportion to the bradycardia, and total peripheral resistance (TPR) is increased so that mean arterial blood pressure remains almost unchanged [7]. Because the increase in peripheral vascular tone during the diving response is sympathetically mediated, it is hypoth*Corresponding author. Tel.: 11-630-515-6386; fax: 11-630-9716414. E-mail address: [email protected] (P.F. McCulloch). 0006-8993 / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03051-8

esized that brainstem pre-sympathetic nuclei are activated during voluntary underwater submergence. ¨ and Fuxe [13] identified numerous groups of Dahlstrom catecholaminergic neurons within the brains of rats. Noradrenergic areas were identified in the medulla and pons as A1 through A7, while adrenergic areas were identified as C1–C3. Many of these brainstem catecholaminergic groups, such as C1 and A5, are important in the control of blood pressure [14,22,63], especially during reflexly produced changes in sympathetic tone. Therefore, the objective of this research was to determine whether catecholaminergic brainstem neurons are activated during the diving response. To investigate this objective, we used a previously developed model of voluntarily diving rats [35,40]. All rats, including laboratory rats, have an innate ability to

P.F. McCulloch, W.M. Panneton / Brain Research 984 (2003) 42–53

43

swim [59], and exhibit the physiological responses to diving typical of small mammals [40]. Thus the brains of rats trained to voluntarily dive underwater were immunohistochemically reacted for both Fos, the protein product of the c-fos gene, as a marker of neuronal activation [6], and the enzyme tyrosine hydroxylase (TH) as a marker of catecholaminergic neurons. Therefore co-expression of both TH and Fos identified an activated catecholaminergic neuron. We show that in voluntarily diving rats, the percentage of Fos and TH double labeled neurons were significantly greater in A1, C1, A2, A5, sub-coeruleus, and globosa neurons, but not in C2 or A7, when compared with Control animals. Portions of this data have been presented previously [36–38].

2. Materials and methods All protocols involving animals were approved by the animal care and use committees of both Midwestern University and Saint Louis University, and adhere to NIH guidelines.

2.1. Dive training Male Sprague–Dawley rats (N512) were obtained commercially (Harlan, Indianapolis, IN, USA) and trained to swim and dive through a maze constructed of Plexiglass 姠 (Fig. 1). Water temperature in the maze was maintained at 3062 8C to reduce thermal stress due to cold-water immersion [33]. The rats were first trained to negotiate a 5 m maze by swimming on the surface of the water. They were next trained to dive underwater through the maze. When placed in the start area, the rats voluntarily initiated their own dives to reach the finish area. The rats appeared unstressed by either the training protocol or exposure to water. No external reward (i.e., food) was used during the training protocol. Rats started the training protocol just after weaning and were housed three to a cage. The rats were trained daily for approximately 5 weeks. After training was complete, the rats were randomly split into three groups on the day of the experiment; one rat per cage was assigned to each of the three groups. The Diving group repetitively dived through the maze every 5 min for 2 h for a total of 24 dives per rat. The Swimming group similarly negotiated the maze every 5 min for 2 h, but swam through the maze without submerging under water. The Control group neither dived nor swam, but remained in their cage for the entire experiment. In total, each group had four rats.

2.2. Immunohistochemical processing One hour after the experiment, the rats were deeply anesthetized (pentobarbital sodium, i.p.) and then perfused

Fig. 1. Schematic drawing of the Plexiglas姠 tank (100360315 cm) used to train rats to voluntarily swim and dive. Vertical pieces inserted into the tank create a simple maze consisting of five channels. The tank is filled with water, and rats are initially trained to negotiate the maze by swimming on the surface of the water. Gradually rats learn to swim from the start area (at upper left), to the finish area (a raised platform at the bottom right). Then the rats are trained to dive through the maze, kept underwater by horizontal Plexiglas姠 pieces placed slightly below the water surface. The underwater length of the maze is approximately 5 m.

transcardially with 0.1 M phosphate-buffered saline (PBS; pH 7.3) followed by 4% paraformaldehyde in PBS. The brains were removed and refrigerated overnight in fixative containing 20% sucrose, and then sectioned at 50 mm on a freezing microtome. To minimize differences in immunohistochemical procedures (i.e., slight differences in reaction times and / or dilution factors of the primary or secondary antibodies) the immunohistochemical processing consisted of tissue from one animal of each of the three groups (Diving, Swimming and Control) that were housed in the same cage. From each brain, every fourth section was processed immunohistochemically with antibodies against both Fos and tyrosine hydroxylase (TH). Sections were initially rinsed in a 1% sodium borohydride solution for 15 min, washed, and then incubated overnight at room temperature with a cocktail of antibodies against Fos (rabbit polyclonal IgG for c-fos p62; 1:1000; Santa Cruz) and TH (mouse IgG specific for TH; 1:5000; DiaSorin) mixed in 0.1 M phosphate buffer with 0.2% Triton. On the following day, the sections were washed and then incubated for 1 h in goat anti-rabbit biotinylated secondary IgG (1:200; Vector). After another washing, the sections were then incubated in an avidin–biotin–peroxidase complex (ABC, Vectastain Elite; Vector). The Fos antigen was visualized in the brainstem with the chromogen diaminobenzidine (DAB) enhanced with nickel ammonium sulfate. After labeling for

44

P.F. McCulloch, W.M. Panneton / Brain Research 984 (2003) 42–53

Fos, the sections were rinsed in Avidin-D and then Dbiotin to reduce cross reactivity in the second stage of the labeling procedure. The sections were then soaked in horse anti-mouse biotinylated secondary IgG (1:200; Vector) for 1 h, incubated in the ABC complex for another hour, and then reacted with DAB solution without nickel ammonium sulfate. Sections were mounted serially on gelatin-coated slides, dehydrated in alcohol, defatted in xylene and coverslipped. Reactive neurons were visualized in a brightfield microscope (Nikon E800); Fos positive neurons appeared as cells with black-labeled nuclei while TH positive neurons had a brown cytoplasm. Double-labeled neurons showed both properties. Profiles of catecholaminergic neurons were counted bilaterally in the A1, A2, C1, C2, C3, A5, sub-coeruleus and A7 regions of the brainstem [49]. Counts were also made of Fos1TH double-labeled neurons. We considered these counts as estimates since they were not stereologically unbiased [10,21]. TH-positive neurons in A6 (locus coeruleus) were not counted because their extremely tight packing density made it impossible to distinguish individual neurons. Instead counts were made of only Fos-positive neurons within A6, with the assumption that every Fos-positive neuron here would also be a catecholaminergic neuron. Representative photomicrographs were taken digitally (Qimaging), saved in the computer and adjusted for color with Adobe PhotoShop.

2.3. Data analysis Prior to data analysis, the slides containing the brainstem sections were given a random code, thus blinding the observer as to which group the brain tissue originated. After the counts were completed, the code was broken and the neuronal counts were tabulated into Control, Swimming and Diving groups, and analyzed statistically using computer software (SigmaStat). Counts of TH-positive neurons and Fos1TH double-labeled neurons were used to estimate the percentage of double-labeled neurons for each brain region. The percentages of double-labeled neurons for each of the rats from the three groups (Control, Swimming and Diving) were averaged to create a grand mean6standard error (S.E.). For each catecholaminergic brainstem region analyzed, one-way analyses of variance (ANOVAs) compared the percentage of double-labeled neurons between the Control, Swimming and Diving groups to determine if diving behavior induced doublelabeling of catecholaminergic neurons. Because we were unable to determine the number of TH-positive neurons within A6, we were unable to determine the percentage of double-labeled neurons. We instead compared the number of Fos-positive neurons per section for A6 between the Control, Swimming and Diving groups. The level of significance was set at P,0.05, and Tukey’s Test was used for post-hoc multiple comparisons to determine significance between groups.

3. Results

3.1. Dive training All rats successfully completed the swim and dive training (Fig. 2). The four rats assigned to either the Swimming or Diving groups completed each of their 24 dives within the 2 h period. The Diving rats took an average of 11.960.5 s (range: 9.6 to 19.0 s) to dive through the maze while the swimming rats took significantly longer, 17.262.2 s (range: 12.4 to 31.5 s), to swim through the maze. The swimming rats often stopped swimming and held onto the walls of the maze, whereas the diving rats progressed underwater through the maze without stopping. Additionally, many rats, while waiting on the rest platform between trials during the training procedure, would reenter the water and start to swim on their own accord, and / or submerge their heads and look around under the water.

3.2. Fos and TH immunohistochemistry All cases resulted in the immunostaining of neurons for TH throughout the brainstem. However, since sections were reacted only with antibodies against TH and not phenylethanolamine-N-methyl transferase (PNMT), no distinction could be made between the subsets of adrenergic and noradrenergic neurons. Thus we could not differentiate between A1 and C1 neurons in the ventrolateral medulla, nor between A2 and C2 neurons in the nucleus tractus solitarii (NTS). However, we defined the catecholaminergic neurons in the ventrolateral medulla as A1 if found caudal to 1200 mm caudal to the caudal pole of the facial nucleus, or as C1 if found rostral to this [44,50]. TH-positive neurons in the NTS were designated as either A2 caudal to the obex or C2 rostral to the obex. THpositive neurons were considered as small (9–13 mm), medium (15–20 mm), or large (22–25 mm), and qualitative estimates of their staining intensity are given below.

3.3. Description of TH-positive neurons In the medulla, A1 neurons were small to medium sized, were moderately stained, and had multipolar reticular shapes. A1 neurons were found near the dorsal and lateral borders of the lateral reticular nucleus (LRN) caudal to the obex, and between the two rostral poles of the LRN just rostral to the obex (Fig. 3A and B). C1 neurons included those ventral to the nucleus ambiguous (up to 1200 mm caudal to the caudal pole of the facial nucleus), and just medial to the caudal facial nucleus (Fig. 3C and D). A2 neurons were generally round, medium sized, and had moderate staining; many had two labeled dendrites (Fig. 5A and B). C2 neurons generally were fewer in number, smaller, more lightly stained, and more reticular in shape than A2 neurons. The reticular, medium-sized neurons of

P.F. McCulloch, W.M. Panneton / Brain Research 984 (2003) 42–53

45

Fig. 2. Training of the rats to swim and dive through the Plexiglas maze. (A) Rats are first trained to swim through the maze on the surface of the water. (B) Rats are then gradually trained to dive underwater through the entire maze, from start area (in upper right) to the finish area (in the bottom left). (C) Close-up of a rat diving underwater. Horizontal Plexiglas姠 pieces prevent the rat from surfacing while diving through the maze. (D) Between trials rats rest in the finish area and groom their wet fur. See Fig. 1 for description of the dive tank.

moderately stained C3 were found near the dorsal midline of the medulla between the hypoglossal and facial motor nuclei; many were interstitial to the fibers of the medial longitudinal fasciculus. In the pons, A5 neurons were generally larger and more densely stained than those seen in more caudal groups (Fig. 3E and F). Neurons were identified as A5 if they were immediately medial to either the subnucleus oralis of the spinal trigeminal complex near the rostral pole of the facial nucleus, or the exiting facial or trigeminal motor roots. Most A5 neurons had spindle or pyramidal shapes, and generally were oriented along a dorsomedial to ventrolateral axis. Labeled catecholamine neurons found ventral to A6 in the pontine central grey and extending

medial and ventral to the trigeminal motor nucleus collectively were considered as sub-coeruleus (Fig. 4A and B). Most sub-coeruleus neurons were medium sized and reticular in shape. However, they were more lightly stained, which contrasted them from the densely stained A5 neurons. Nevertheless, catecholamine neurons ventral to the trigeminal motor nucleus interfaced with the rostral pole of the dorsal A5, and these neurons were placed in either sub-coeruleus or A5 with much difficulty. At levels rostral to the trigeminal motor nucleus, immunopositive neurons in positions similar to sub-coeruleus were considered A7 (Fig. 4C and D). A7 neurons were medium-sized and were very lightly stained, similar to sub-coeruleus. These were contrasted with the large darkly stained

46

P.F. McCulloch, W.M. Panneton / Brain Research 984 (2003) 42–53

Fig. 3. Photomicrographs of brain tissue immunohistologically processed for both Fos and tyrosine hydroxylase (TH) from the medullas of Control and Diving rats. Immunohistological processing produced brown TH somas and black Fos nuclei. Single-labeled TH-positive neurons are indicated by open arrowheads, while Fos1TH double-labeled neurons are indicated by solid arrows. Large arrows and open arrowheads point to neurons shown in insets. A1 neurons from (A) a Control rat and (B) a Diving rat. C1 neurons from (C) a Control rat and (D) a Diving rat. A5 neurons from (E) a Control rat and (F) a Diving rat. More Fos and TH double-labeled neurons can be seen in the A1, C1 and A5 regions of the Diving rat, compared with the corresponding Control rat. Abbreviations: cc, central canal; 7n, Facial nerve; SO, superior olive. Calibration bar in E is for panels A–F, and is 250 mm. Calibration bar in inset in F is for all insets, and is 50 mm.

globosa neurons [39] that were adjacent to the lateral lemniscus and had processes extending laterally into the white matter (Fig. 4E and F).

3.4. Fos1 TH double-labeling in the medulla There were few Fos labeled catecholaminergic neurons in the brainstems of Control rats except for A2, which had 8.664.2% double-labeled neurons (Table 1). Although C3 was labeled with TH in brains from all groups, it showed no evidence of Fos, and no double-labeled neurons were seen there in any of the three groups. There was a significant increase in the percentage of Fos1TH double-labeled neurons of A1 in both Swimming and Diving rats compared with Control rats (Fig. 3A and B, and Table 1). Moreover, there was a significantly greater percentage of double-labeled A1 neurons in Diving rats compared to Swimming rats. In C1 of Diving rats

there was a significant increase in the percentage of double-labeled neurons compared with both the Control and Swimming rats (Fig. 3C and D, and Table 1). In addition there was a significant increase in the percentage of Fos1TH double-labeled neurons in the Diving rats compared with the Swimming rats. In A2 there was a significant increase in the percentage of Fos1TH neurons in the Diving rats compared with the Control rats (Fig. 5A and B, and Table 1). In C2 there were no significant differences in the percentage of Fos1TH double-labeled neurons between the Control, Swimming or Diving rats (Table 1).

3.5. Fos1 TH double-labeling in the pons In A5, less than 1% of the neurons in the Control animals were double-labeled (Fig. 3E and F, and Table 1).

P.F. McCulloch, W.M. Panneton / Brain Research 984 (2003) 42–53

47

Fig. 4. Photomicrographs of brain tissue immunohistologically processed for both Fos and tyrosine hydroxylase (TH) from the pons of Control and Diving rats. Immunohistological processing produced brown TH somas and black Fos nuclei. Single-labeled TH-positive neurons are indicated by open arrowheads, while Fos1TH double-labeled neurons are indicated by solid arrows. Large arrow and open arrowheads point to neurons shown in insets. Very few double-labeled neurons were found in sub-coeruleus in either the (A) Control or (B) Diving rat, although significantly more double-labeled neurons were observed in Diving rats. Note that TH labeling in sub-coeruleus tended to be very light. Very few double-labeled A7 neurons were found in either the (C) Control or (D) Diving rat. Globosa neurons from a (E) Control and (F) Diving rat. These globosa neurons were morphologically distinct, were located more laterally to the spinocerebellar tract, and were always darkly labeled with TH. Note that in the Control rat the globosa neurons were not double-labeled, but that in the Diving rat most globosa neurons were double-labeled. Stars in C and D identify location of globosa neurons. Stars in E and F identify A7 location. Abbreviation: MoV, trigeminal motor nucleus. Calibration bar in E is for panels A–F, and is 250 mm. Calibration bar in inset in F is for all insets, and is 50 mm.

In contrast, 39 and 65% of A5 neurons were doublelabeled in the Swimming and Diving animals, respectively. Moreover, the percentage of double-labeled A5 neurons Table 1 Percentage of Fos and TH double-labeled neurons (mean6S.E.) in brainstem catecholaminergic regions Brain region

Control

Swimming

Diving

A1 C1 A2 C2 A5 Sub-coeruleus A7 Globosa neurons

0.760.6 0.660.4 8.664.2 1.061.0 0.560.3 0 0 1.161.1

39.763.1 1 14.465.1 24.8610.1 13.969.2 38.663.2 1 8.661.1 1 3.161.8 52.4610.2 1

66.966.4 1,2 49.568.1 1,2 53.4613.2 1 16.7616.7 64.969.3 1,2 12.862.7 1 5.963.4 80.6612.5 1

15Significantly different from control; 25significantly different from swimming.

was significantly greater in Diving rats compared with Swimming rats. There was a significant increase in the percentage of double-labeled neurons in sub-coeruleus in both the Swimming and Diving groups compared with the Control group (Fig. 4A and B, and Table 1). However, the total percent of Fos1TH double-labeled neurons was small. The statistical increase in the percentage of Fos1 TH double-labeled neurons in sub-coeruleus may have been due to the strict criteria used in assigning a Fos1TH double-labeled neuron to a particular subgroup (see below). There was no significant increase in the percentage of double-labeled A7 neurons in the Swimming and Diving groups (Fig. 4C and D, and Table 1). It was of interest that the greatest percentage increase of double-labeled neurons in the lower brainstem of Swimming and Diving rats versus Controls was found in the large darkly-stained globosa neurons (see Ref. [39]), which were located lateral

48

P.F. McCulloch, W.M. Panneton / Brain Research 984 (2003) 42–53

Fig. 5. Photomicrographs of brain tissue immunohistologically processed for both Fos and tyrosine hydroxylase (TH). Single-labeled TH-positive neurons are indicated by open arrowheads, while Fos1TH double-labeled neurons are indicated by solid arrows. Large arrow and open arrowhead point to neurons shown in insets. A2 neurons from (A) a Control rat and (B) a Diving rat. More Fos and TH double-labeled neurons can be located the A2 region of the Diving rat, compared with the corresponding Control rat. A6 neurons from (C) a Control rat and (D) a Swimming rat. TH-positive neurons in A6 were not counted because of their tight packing density. Instead, counts were made only of Fos-positive neurons within A6. The number of Fos-positive neurons per section was increased in the Swimming rat compared with the Control rat. The calibration bar in C is for panels A–D, and is 250 mm. Calibration bar in inset in B is for both insets, and is 50 mm.

to the main grouping of A7 neurons (Fig. 4E and F, and Table 1). Over 80% of the globosa neurons in the Diving rats, and 52% of the globosa neurons in the Swimming rats were Fos-positive, which contrasts with the 1% found in Control rats. In A6 of the Control rats there were 4.261.2 Fospositive neurons per section (Fig. 5C and D). The number of Fos-positive neurons per section increased in both the Swimming (23.764.0) and Diving (21.467.0) rats. The Fos labeling was especially prominent towards the dorsal half of A6. However, the increase in Fos labeling was only significant for the Swimming rats compared with the Control rats. The number of A6 Fos-positive neurons per section in the Swimming and Diving rats were not significantly different from each other.

4. Discussion This study shows that laboratory rats can be trained to voluntarily dive underwater with little apparent stress. Moreover, the labeling of brainstem catecholaminergic neurons with Fos shows that a significant percentage of these neurons are activated during diving, as compared with either swimming or control activity. A1, C1, A2, A5 and globosa neurons all showed a significant increase in Fos1TH double-labeling in Diving versus Control animals. A1, C1, and A5 showed a significant increase in double-labeling in Diving versus Swimming rats. Also, there were significant increases in Fos1TH double-labeled neurons in A1, A5, sub-coeruleus and globosa neurons in Swimming rats compared with Control rats. In contrast, the

P.F. McCulloch, W.M. Panneton / Brain Research 984 (2003) 42–53

catecholaminergic neurons of A7 and C2 did not show a significant increase in double labeling, and C3 neurons were never double-labeled. Finally, there was a significant increase in Fos-positive neurons in A6 of Swimming rats, but not Diving rats, compared with Control rats.

4.1. Dive training Rats are often regarded as strictly terrestrial animals. However, feral Rattus norvegicus often live in wet places, such as sewers, rivers, ditches and marshes [24]. Rats are excellent swimmers and often dive to use underwater entrances to their dens [24]. The laboratory rat is descended from the semi-aquatic wild rat, having been colonized for research purposes around 150 years ago. In addition, laboratory rats exhibit the typical mammalian cardiovascular and respiratory responses to diving [40,48]. McCulloch et al. [40] showed that in voluntarily diving rats, heart rate decreases by 83% (478613 to 83616 beats / min) immediately upon submersion and remains at this level for the duration of the dive. Cardiac output decreases in proportion to the bradycardia. However, arterial blood pressure is generally maintained at pre-dive values, primarily because peripheral resistance is increased 437–463% throughout the dive [35]. The cardiovascular changes associated with voluntary diving in rats only occur after submersion of the nose under water [35]. The Swimming rats swam through the maze on the surface of the water, and therefore did not experience the sensation of underwater submergence. However, the Swimming rats experienced some of the same sensory inputs (i.e., proprioceptive feedback from exercising muscles, body immersion in water, and / or thermal sensation from the water) as the Diving rats. These sensory inputs, and possibly the cardiovascular responses to exercise due to the swimming, may explain why the Swimming rats had increased Fos1TH double labeling in the A1, C1, A5, sub-coeruleus and globosa neurons, compared to the Control rats. It was only the Diving rats, however, that experienced repetitive underwater submersion. There were significant increases in the number of double-labeled Fos1TH neurons between the Swimming and Diving rats in the A1, C1 and A5 areas. This indicates that underwater submergence activates additional catecholaminergic neurons compared to just swimming on the surface of the water. Presumably, initiation and central integration of the diving response accounts for the activation of these additional catecholaminergic neurons in the brainstems of the Diving rats compared with the Swimming rats, and suggests that neurons in these groups may be part of the central circuit of the diving response. Finally, in both the Diving and Swimming rats, there were significant increases in Fos1 TH labeling in sub-coeruleus and globosa neurons compared with Control rats. The activation of these neurons during swimming does not preclude them as integral to the

49

central circuitry of the diving response. They may instead be important for the cardiovascular, somatosensory and / or proprioceptive responses to both swimming and diving. In A6 there was an increase in Fos-positive neurons in both the Swimming and Diving rats compared with the Control rats, although the increase in Fos-labeling was only significantly different between the Swimming and Control rats. The locus coeruleus (LC) (A6) is thought to play a role in enhanced vigilance, arousal and / or attention to sensory stimuli [1,3], as prominent or novel stimuli in the external environment increase the discharge rate of LC neurons [2]. During the 2 h of repetitive activity, both Swimming and Diving rats experienced similar environmental stimuli, including human handling, compared with the Control rats that were left untouched in their cages. It is possible that the increase in both environmental stimuli and activity levels during Diving or Swimming trials activated neurons within the LC [1]. Additionally, the cutaneous sensation of water immersion experienced by the Swimming and Diving rats may also have activated LC neurons [17]. The LC may also play a role in cardiovascular control [30], although LC neurons seem to be regulated by low pressure blood volume receptors rather than by high pressure arterial baroreceptors [16,18], and in the present study blood volume should not have been affected by swimming or diving activity. In any event, there was similar activation of LC neurons in Swimming and Diving rats, indicating that underwater submersion was no greater a stressor to the rats than was swimming on the surface of the water.

4.2. Technical considerations c-fos is an immediate early gene thought to participate in the alteration of long-term cellular function [46]. Immunohistological detection of Fos, the protein product of the c-fos gene, is often used as a marker of neuronal activation [6]. As such, Fos expression potentially represents a powerful metabolic marker for tracing medullary neuronal circuits [15], including the brainstem circuitry of the cardiorespiratory response to diving. The Fos technique does have limitations, however. For instance, production of the Fos protein indicates that a neuron has been activated, but it does not indicate the function of that neuron. Any neuron that is part of a reflex circuit could have an afferent, efferent, or integrative function. Additionally, Fos may not be expressed equally in all neurons [15]. For instance, neurons that are inhibited as part of a dynamic circuit will not express Fos [41]. Therefore, the Fos technique can only be used to identify neurons that have been activated during repetitive stimulation, but cannot be used to identify all the brainstem neurons that are part of a dynamic cardiorespiratory circuit. However, neurons that do express Fos and TH after repetitive voluntary diving presumably are catecholaminergic neurons activated during underwater submerg-

50

P.F. McCulloch, W.M. Panneton / Brain Research 984 (2003) 42–53

ence. Therefore a Fos-labeled catecholaminergic region of Diving rats could be part of the brainstem circuitry of the diving response. Additionally, our data indicate that the Control rats that remained in their cages produced very little Fos immunoreactivity in catecholaminergic neurons.

4.3. Fos1 TH double-labeling in the medulla Many of the catecholaminergic areas that were activated during diving are involved in cardiovascular autonomic functioning. For instance, adrenergic C1 neurons of the rostral ventrolateral medulla (RVLM) play a crucial role in the tonic and phasic regulation of blood pressure (for reviews, see Refs. [14,22,23]). The RVLM is the most important of the five centrally located presympathetic nuclei [14], and is an important source of tonic excitatory drive to sympathetic vasomotor neurons controlling peripheral vasculature [22,23]. RVLM neurons are also a critical component of central cardiovascular reflex pathways, and function as the efferent limb of the vasomotor component of the arterial baroreflex [14,22,63]. Additionally, neurons within the RVLM are important in producing the increase in peripheral vasoconstriction during nasal stimulation in anesthetized rats [42]. Nearly 62% of barosensitive RVLM neurons increased their firing rate during nasal stimulation, even with a concomitant increase in arterial blood pressure. Approximately 60% of all bulbospinal RVLM neurons are catecholaminergic C1 neurons [44,67], and C1 cells may be critical for the full expression of sympathoexcitatory responses generated by the RVLM [57,58]. Therefore, in the Diving rats, the activation of C1 neurons we describe could be important for the increase in peripheral vasoconstriction observed during voluntary diving [40]. The A1 group sends projections to the nucleus tractus solitarius, central gray, magnocellular neurosecretory neurons in the hypothalamus, but not to the spinal cord [25,43,47,56,71]. The noradrenergic A1 neurons participate in the physiological response to hypotension, and appear to be essential links in the central pathway mediating the baroreceptor-initiated secretion of vasopressin [27,28,60–62]. Decreasing arterial pressure activates A1 cells [5,20]. During diving in rats, there is often an initial decrease in arterial pressure immediately upon submersion [40]. This is due to the enormous parasympatheticallymediated decrease in cardiac output [35]. However, a few seconds into the dive the sympathetically-mediated increase in peripheral resistance matches or exceeds the decrease in cardiac output, and arterial blood pressure then returns to, or above, pre-dive levels [35,40]. In the present study we found that 66% of the A1 neurons were activated during repetitive diving. In comparison, Gieroba et al. [19] found that 18% of A1 neurons are activated when formaldehyde vapors are passed through the nasal passage of anesthetized rabbits to initiate an autonomic reflex similar to that of the diving response. It may be possible that

arterial baroreceptors were stimulated during diving by the initial drop in arterial pressure, thus activating the noradrenergic A1 neurons in response to the transient hypotension. Alternatively, because the A1 Fos labeling was increased in both swimming and diving animals compared with the control animals, it may be possible that activation of A1 occurred in response to repetitive exercise (either swimming or diving), rather than to submersion-induced hypotension. Little is known about the function of the catecholaminergic neurons in the nucleus tractus solitarius, the A2 and C2 groups. They do not receive a significant baroreceptor input [45], but they do project rostrally to the periaqueductal gray [25], forebrain, hypothalamus and amygdala [11,12,51,55], as well as caudally to the spinal cord [55]. However, over 50% of the A2 neurons were double labeled in the Diving rats. The role of these neurons in diving rats is presently unknown.

4.4. Fos1 TH double-labeling in the pons A5 neurons project to the intermediolateral cell column of the spinal cord [8,32,65,66], and provide an important noradrenergic input to sympathetic preganglionic neurons [70]. The cardiovascular role of A5 neurons primarily is excitatory [26,31], although A5 appears to have little tonic influence on resting sympathetic nerve activity [34]. A5 neurons play a role in the sympathetic response to arterial chemoreceptor stimulation [29]. Repetitive diving activated nearly two-thirds of all the A5 neurons, and therefore these neurons may also play a role in the sympathetic response to underwater diving. A7 and sub-coeruleus neurons project to the spinal cord [9,53,54,64,68–70], and appear to be involved in modulating nociception (reviewed by Bajic and Proudfit [4]). It is of interest that few neurons of either group were double labeled with Fos and TH during the present experiments in diving rats. If the function of these pontine catecholaminergic neurons is indeed antinociceptive, it would suggest they are inactive during diving in awake voluntarily diving rats. This would also suggest that the receptors initiating the cardiorespiratory responses observed during diving are not responding to painful stimuli. The catecholaminergic neurons of sub-coeruleus and A7 could generally be distinguished from A5 neurons by the differences in their shape and staining intensity. However, we used anatomical boundaries, rather than neuronal shape or staining intensity, to assign a TH positive neuron to a particular group. Consequently, it is certain that some A5 neurons were counted as either sub-coeruleus or A7. Although the absolute numbers of such neurons are small, this may have resulted in sub-coeruleus labeling being significantly increased in the Swimming and Diving rats, compared to the Control rats. In any event, sub-coeruleus probably is not directly involved in central cardiovascular

P.F. McCulloch, W.M. Panneton / Brain Research 984 (2003) 42–53

functioning, but rather more likely modulates nociceptive or motor functioning [52]. An interesting finding of the present study was the very prominent double-labeling of globosa neurons located in the lateral pons [39]. Globosa neurons are a morphologically distinct subgroup of A7 neurons, being larger, more globularly shaped, and having dendritic processes that are oriented transversely and extend into the lateral lemniscus. In addition, globosa neurons stain more prominently for TH than do the main grouping of A7 neurons [39]. The expression of Fos in the globosa neurons was significantly increased in both Swimming and Diving rats compared with Control rats, and in the diving animals, 80% of the globosa neurons had been activated. In comparison, very few A7 or sub-coeruleus neurons were activated during diving. This suggests that the globosa neurons are functionally distinct from the main grouping of A7 neurons, and are selectively activated during swimming and diving. At present the function of globosa neurons is unknown. However electrical stimulation of the A7 group produces a significant increase in mean arterial pressure [72], a response that is also observed in voluntarily diving rats [40]. It therefore may be possible that globosa neurons help produce the increase in peripheral vasoconstriction during underwater submergence.

4.5. Fos-labeling in non-TH-positive neurons Fos-positive neurons were also noted in many noncatecholaminergic regions. For instance, an increase in the number of Fos-positive neurons have been reported in the trigeminal medullary dorsal horn in diving rats [36], which may represent the afferent limb of the reflex circuit. Additionally, Fos-positive and TH-negative neurons were found in most of the catecholminergic regions described herein (i.e., A1, C1 and A5 in Fig. 3, A7 in Fig. 4, and A2 in Fig. 5). Numerous Fos-positive neurons were seen throughout the rostrocaudal extent of the ventrolateral medulla, which contains abundant non-catecholaminergic cardiovascular and respiratory neurons [14,22,63]. Neurons with cardiovascular and respiratory functions are also found in the NTS and parabrachial complex, and many neurons labeled only with Fos were found in these regions. The distribution of these non-catecholaminergic neurons containing Fos will be reported in the future. For instance, it is likely that non-catecholaminergic neurons, i.e., cardiac vagal motorneurons and some neurons with respiratoryrelated activity, would have been activated during the underwater submergence.

5. Conclusions In conclusion, we show that underwater submersion produces activation of catecholaminergic neurons within the brainstem of rats. Additionally, this catecholaminergic

51

activation is selective, with increase in Fos1TH doublelabeling occurring in A1, C1, A2, A5, subcoeruleus and globosa neurons, but not in C2, C3 or A7. We suggest that the activation of C1 and A5 neurons may be involved in producing the increase in sympathetic tone that occurs during diving.

Acknowledgements Supported by an American Heart Association, Missouri Affiliate, Grant-in-Aid 9806384 and NIH grant HL67045 to P.F.M., and NIH grants HL38471 HL64772 to W.M.P.

References [1] G. Aston-Jones, J. Rajkowski, J. Cohen, Role of locus coeruleus in attention and behavioral flexibility, Biol. Psychiat. 46 (1999) 1309– 1320. [2] G. Aston-Jones, J. Rajkowski, P. Kubiak, T. Alexinsky, Locus coeruleus neurons in monkey are selectively activated by attended cues in a vigilance task, J. Neurosci. 14 (1994) 4467–4480. [3] G. Aston-Jones, M.T. Shipley, R. Grzanna, The locus coeruleus, A5 and A7 noradrenergic cell groups, in: G. Paxinos (Ed.), The Rat Nervous System, Academic Press, Sydney, 1995, pp. 183–213. [4] D. Bajic, H.K. Proudfit, Projections of neurons in the periaqueductal gray to pontine and medullary catecholamine cell groups involved in the modulation of nociception, J. Comp. Neurol. 405 (1999) 359– 379. [5] W.W. Blessing, J.O. Willoughby, Inhibiting the rabbit caudal ventrolateral medulla prevents baroreceptor-initiated secretion of vasopressin, J. Physiol. (Lond.) 367 (1985) 253–265. [6] E. Bullitt, Expression of C-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat, J. Comp. Neurol. 296 (1990) 517–530. [7] P.J. Butler, D.R. Jones, The comparative physiology of diving in vertebrates, in: O. Lowenstein (Ed.), Advances in Comparative Physiology and Biochemistry, Vol. 8, Academic Press, New York, 1982, pp. 179–364. [8] C.E. Byrum, R. Stornetta, P.G. Guyenet, Electrophysiological properties of spinally-projecting A5 noradrenergic neurons, Brain Res. 303 (1984) 15–29. [9] F.M. Clark, H.K. Proudfit, The projection of noradrenergic neurons in the A7 catecholamine cell group to the spinal cord in the rat demonstrated by anterograde tracing combined with immunocytochemistry, Brain Res. 547 (1991) 279–288. [10] R.E. Coggeshall, H.A. Lekan, Methods for determining numbers of cells and synapses: a case for more uniform standards of review, J. Comp. Neurol. 364 (1996) 6–15. [11] E.T. Cunningham Jr., M.C. Bohn, P.E. Sawchenko, Organization of adrenergic inputs to the paraventricular and supraoptic nuclei of the hypothalamus in the rat, J. Comp. Neurol. 292 (1990) 651–667. [12] E.T. Cunningham Jr., P.E. Sawchenko, Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat hypothalamus, J. Comp. Neurol. 274 (1988) 60–76. ¨ K. Fuxe, Evidence for the existence of monoamine[13] A. Dahlstrom, containing neurons in the central nervous system, Acta Physiol. Scand. 62 (Suppl. 232) (1964) 1–55. [14] R.A.L. Dampney, Functional organization of central pathways regulating the cardiovascular system, Physiol. Rev. 74 (1994) 323– 364.

52

P.F. McCulloch, W.M. Panneton / Brain Research 984 (2003) 42–53

[15] M. Dragunow, R. Faull, The use of c-fos as a metabolic marker in neuronal pathway tracing, J. Neurosci. Methods 29 (1989) 261–265. [16] M. Elam, T.H. Svensson, P. Thoren, Differentiated cardiovascular afferent regulation of locus coeruleus neurons and sympathetic nerves, Brain Res. 358 (1985) 77–84. [17] M. Elam, T.H. Svensson, P. Thoren, Locus coeruleus neurons and sympathetic nerves: activation by cutaneous sensory afferents, Brain Res. 366 (1986) 254–261. [18] M. Elam, T. Yao, T.H. Svensson, P. Thoren, Regulation of locus coeruleus neurons and splanchnic, sympathetic nerves by cardiovascular afferents, Brain Res. 290 (1984) 281–287. [19] Z.J. Gieroba, Y.-H. Yu, W.W. Blessing, Vasoconstriction induced by inhalation of irritant vapour is associated with appearance of Fos protein in C1 catecholamine neurons in rabbit medulla oblongata, Brain Res. 636 (1994) 157–161. ´ [20] R.E. Gomez, M.A. Cannata, T.A. Milner, M. Anwar, D.J. Reis, D.A. Ruggiero, Vasopressinergic mechanisms in the nucleus reticularis lateralis in blood pressure control, Brain Res. 604 (1993) 90–105. [21] R.W. Guillery, On counting and counting errors, J. Comp. Neurol. 447 (2002) 1–7. [22] P.G. Guyenet, Role of the ventral medulla oblongata in blood pressure regulation, in: A.D. Loewy, K.M. Spyer (Eds.), Central Regulation of Autonomic Function, Oxford University Press, New York, 1990, pp. 145–167. [23] P.G. Guyenet, N. Koshiya, D. Huangfu, S.C. Baraban, R.L. Stornetta, Y.-W. Li, Role of medulla oblongata in generation of sympathetic and vagal outflows, in: G. Holstege, C.B. Saper (Eds.), Progress in Brain Research, Elsevier, New York, 1996, pp. 127–144. [24] P.W. Hanney, Rodents: Their Lives and Habits, David and Charles, Vancouver, 1975. [25] H. Herbert, C.B. Saper, Organization of medullary adrenergic and noradrenergic projections to the periaqueductal gray matter in the rat, J. Comp. Neurol. 315 (1992) 34–52. [26] D.H. Huangfu, N. Koshiya, P.G. Guyenet, A5 noradrenergic unit activity and sympathetic nerve discharge in rats, Am. J. Physiol. Reg. (Int. Comp. Physiol. 30) 261 (1991) R393–R402. [27] H. Kawano, S. Masuko, Synaptic contacts between nerve terminals originating from the ventrolateral medullary catecholaminergic area and median preoptic neurons projecting to the paraventricular hypothalamic nucleus, Brain Res. 817 (1999) 110–116. [28] H. Kawano, S. Masuko, Tyrosine hydroxylase-immunoreactive projections from the caudal ventrolateral medulla to the subfornical organ in the rat, Brain Res. 903 (2001) 154–161. [29] N. Koshiya, P.G. Guyenet, A5 noradrenergic neurons and the carotid sympathetic chemoreflex, Am. J. Physiol. (Reg. Int. Comp. Physiol. 36) 267 (1994) R519–R526. [30] Y.W. Li, R.A.L. Dampney, Expression of FOS-like protein in brain following sustained hypertension and hypotension in conscious rabbits, Neuroscience 61 (1994) 613–634. [31] A.D. Loewy, E.M. Gregorie, S. McKellar, R.P. Baker, Electrophysiological evidence that the A5 catecholamine cell group is a vasomotor center, Brain Res. 178 (1979) 196–200. [32] A.D. Loewy, S. McKellar, C.B. Saper, Direct projections from the A5 catecholamine cell group to the intermediolateral cell column, Brain Res. 174 (1979) 309–314. [33] R.A. MacArthur, Aquatic thermoregulation in the muskrat (Ondatra zibethicus): energy demands of swimming and diving, Can. J. Zool. 62 (1984) 241–248. [34] D.N. Maiorov, S.C. Malpas, G.A. Head, Influence of pontine A5 region on renal sympathetic nerve activity in conscious rabbits, Am. J. Physiol. (Reg. Int. Comp. Physiol. 278) 278 (2000) R311–R319. [35] P.F. McCulloch, Trigeminal mediation of the initiation of the mammalian diving response. Ph.D. Thesis, University of Saskatchewan, Saskatoon, 1995. [36] P.F. McCulloch, Brainstem neuronal activation during voluntary underwater submergence in rats, Neurosci. Abstr. 25 (1999) 1174. [37] P.F. McCulloch, Trigeminal stimulation produces FOS labeling

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45] [46]

[47]

[48] [49] [50]

[51]

[52] [53]

[54]

[55]

[56]

[57]

within the nucleus tractus solitarius of conscious rats, Physiologist 43 (2000) 263. P.F. McCulloch, Voluntary underwater submergence in conscious rats activates pre-sympathetic brainstem nuclei, Physiologist 45 (2002) 332. P.F. McCulloch, Globosa neurons: a distinct subgroup of noradrenergic neurons in the caudal pons of rats, Brain Res. 964 (2003) 164–167. P.F. McCulloch, G.P. Ollenberger, L.K. Bekar, N.H. West, Trigeminal and chemoreceptor contributions to bradycardia during voluntary dives in rats, Am. J. Physiol. (Reg. Int. Comp. Physiol. 42) 273 (1997) R814–R822. P.F. McCulloch, W.M. Panneton, FOS immunohistochemical determination of brainstem neuronal activation in the muskrat after nasal stimulation, Neuroscience 78 (1997) 913–925. P.F. McCulloch, W.M. Panneton, P.G. Guyenet, The rostral ventrolateral medulla mediates the sympathoexcitation produced by chemical stimulation of the rat nasal mucosa, J. Physiol. (Lond.) 516 (1999) 471–484. S. McKellar, A.D. Loewy, Efferent projections of the A1 catecholamine cell group in the rat: an autoradiographic study, Brain Res. 241 (1982) 11–29. J. Minson, I. Llewellyn-Smith, A. Neville, P. Somogyi, J. Chalmers, Quantitative analysis of spinally projecting adrenaline-synthesising neurons of C1, C2 and C3 groups in rat medulla oblongata, J. Auton. Nerv. Syst. 30 (1990) 209–220. S.D. Moore, P.G. Guyenet, Effect of blood pressure on A2 noradrenergic neurons, Brain Res. 338 (1985) 169–172. J.I. Morgan, T. Curran, Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun, Annu. Rev. Neurosci. 14 (1991) 421–451. E.P. Mtui, M. Anwar, D.J. Reis, D.A. Ruggiero, Medullary visceral reflex circuits: local afferents to nucleus tractus solitarii synthesize catecholamines and project to thoracic spinal cord, J. Comp. Neurol. 351 (1995) 5–26. G.P. Ollenberger, N.H. West, Distribution of regional cerebral blood flow in voluntarily diving rats, J. Exp. Biol. 201 (1998) 549–558. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1998. J.K. Phillips, A.K. Goodchild, R. Dubey, E. Sesiashvili, M. Takeda, J. Chalmers, P.M. Pilowsky, J. Lipski, Differential expression of catecholamine biosynthetic enzymes in the rat ventrolateral medulla, J. Comp. Neurol. 431 (2001) 20–34. P.M. Plotsky, E.T. Cunningham Jr., E.P. Widmaier, Catecholaminergic modulation of corticotropin-releasing factor and adrenocorticotropin secretion, Endocr. Rev. 10 (1989) 437–458. H.K. Proudfit, F.M. Clark, The projections of locus coeruleus neurons to the spinal cord, Prog. Brain Res. 88 (1991) 123–141. V.K. Reddy, S.J. Fung, H. Zhuo, C.D. Barnes, Spinally projecting noradrenergic neurons of the dorsolateral pontine tegmentum: a combined immunocytochemical and retrograde labeling study, Brain Res. 491 (1989) 144–149. V.K. Reddy, S.J. Fung, H. Zhuo, C.D. Barnes, Localization of enkephalinergic neurons in the dorsolateral pontine tegmentum projecting to the spinal cord of the cat, J. Comp. Neurol. 291 (1990) 195–202. P.E. Sawchenko, M.C. Bohn, Glucocorticoid receptor-immunoreactivity in C1, C2, and C3 adrenergic neurons that project to the hypothalamus or to the spinal cord in the rat, J. Comp. Neurol. 285 (1989) 107–116. P.E. Sawchenko, L.W. Swanson, The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat, Brain Res. Rev. 4 (1982) 275–325. A.M. Schreihofer, P.G. Guyenet, Sympathetic reflexes after depletion of bulbospinal catecholaminergic neurons with anti-DbetaHsaporin, Am. J. Physiol. (Reg. Int. Comp. Physiol. 279) 279 (2000) R729–R742.

P.F. McCulloch, W.M. Panneton / Brain Research 984 (2003) 42–53 [58] A.M. Schreihofer, R.L. Stornetta, P.G. Guyenet, Regulation of sympathetic tone and arterial pressure by rostral ventrolateral medulla after depletion of C1 cells in rat, J. Physiol. (Lond.) 529 (2000) 221–236. [59] S. Shapiro, M. Salas, K. Vokovich, Hormonal effects on ontogeny of swimming ability in the rat: assessment of central nervous system development, Science 168 (1970) 147–152. [60] S. Shioda, M. Iwase, I. Homma, S. Nakajo, K. Nakaya, Y. Nakai, Vasopressin neuron activation and Fos expression by stimulation of the caudal ventrolateral medulla, Brain Res. Bull. 45 (1998) 443– 450. [61] S. Shioda, Y. Nakai, Noradrenergic innervation of vasopressincontaining neurons in the rat hypothalamic supraoptic nucleus, Neurosci. Lett. 140 (1992) 215–218. [62] S. Shioda, Y. Shimoda, Y. Nakai, Ultrastructural studies of medullary synaptic inputs to vasopressin-immunoreactive neurons in the supraoptic nucleus of the rat hypothalamus, Neurosci. Lett. 148 (1992) 155–158. [63] K.M. Spyer, Central nervous mechanisms contributing to cardiovascular control, J. Physiol. (Lond.) 474 (1994) 1–19. ¨ [64] R.T. Stevens, C.J. Hodge Jr., A.V. Apkarian, Kolliker-Fuse nucleus: the principal source of pontine catecholaminergic cells projecting to the lumbar spinal cord of cat, Brain Res. 239 (1982) 589–594. [65] A.M. Strack, W.B. Sawyer, J.H. Hughes, K.B. Platt, A.D. Loewy, A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infection, Brain Res. 491 (1989) 156–162.

53

[66] A.M. Strack, W.B. Sawyer, K.B. Platt, A.D. Loewy, CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labelling with pseudorabies virus, Brain Res. 491 (1989) 274–296. [67] D.C. Tucker, C.B. Saper, D.A. Ruggiero, D.J. Reis, Organization of central adrenergic pathways: I. Relationships of ventrolateral medullary projections to the hypothalamus and spinal cord, J. Comp. Neurol. 259 (1987) 591–603. [68] K.N. Westlund, R.M. Bowker, M.G. Ziegler, J.D. Coulter, Origins of spinal noradrenergic pathways demonstrated by retrograde transport of antibody to dopamine-beta-hydroxylase, Neurosci. Lett. 25 (1981) 243–249. [69] K.N. Westlund, R.M. Bowker, M.G. Ziegler, J.D. Coulter, Descending noradrenergic projections and their spinal terminations, Prog. Brain Res. 57 (1982) 219–238. [70] K.N. Westlund, R.M. Bowker, M.G. Ziegler, J.D. Coulter, Noradrenergic projections to the spinal cord of the rat, Brain Res. 263 (1983) 15–31. [71] J.M. Woulfe, B.A. Flumerfelt, A.W. Hrycyshyn, Efferent connections of the A1 noradrenergic cell group: a DBH immunohistochemical and PHA-L anterograde tracing study, Exp. Neurol. 109 (1990) 308–322. [72] D.C. Yeomans, F.M. Clark, J.A. Paice, H.K. Proudfit, Antinociception induced by electrical stimulation of spinally projecting noradrenergic neurons in the A7 catecholamine cell group of the rat, Pain 48 (1992) 449–461.