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Volume 17, Number 9, Issue of May 1, 1997 pp. 3364-3378
Copyright ©1997 Society for NeuroscienceRole of the Midbrain Periaqueductal Gray in Maternal Nurturance and Aggression: c-fos and Electrolytic Lesion Studies in Lactating Rats
Joseph S. Lonstein and Judith M. Stern Department of Psychology, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903
ABSTRACT
The upright crouched, or kyphotic, nursing posture of lactating rats is dependent on suckling stimulation from pups. Because of the neuroanatomical connections of the periaqueductal gray (PAG) and its sensorimotor integration of the analogous lordosis posture displayed by sexually receptive female rats, the possible role of the PAG in kyphosis was investigated using c-fos immunocytochemistry and electrolytic lesions. Lactating rats interacting with and nursing a litter of suckling pups showed greater Fos-immunoreactive nuclei in the lateral and ventrolateral caudal PAG (cPAGl,vl) compared with dams receiving nonsuckling somatosensory, distal, or no stimulation from pups. In contrast, this pattern was not evident in the rostral PAG, where the highest Fos levels occurred in nonsuckled dams, or in five other brainstem sites with either no group differences (peripeduncular, dorsal raphe, and pontine nuclei) or negligible Fos (ventral tegmental area, spinal trigeminal nuclei). After bilateral electrolytic lesions of the cPAGl,vl during gestation or on day 7 postpartum, active maternal behaviors, such as retrieval and licking of pups, and total nursing time were essentially normal. Kyphotic nursing, however, was reduced by 85%, nursing in prone and supine postures increased substantially, and 24 hr litter weight gains were reduced, particularly early in lactation (by 26%). Furthermore, lesioned rats attacked a strange male twice as often as controls did, which is suggestive of reduced fearfulness. These results extend the known roles of the PAG in reproductive and defensive behaviors to the postural control of suckling-induced kyphotic nursing and the modulation of maternal aggression.
Key words: nursing behavior; maternal aggression; c-fos; suckling; lactation; rostral PAG; caudal PAG
INTRODUCTION
The periaqueductal gray (PAG) is involved in many functions, including vocalization (Waldbillig, 1975
; Holstege, 1989
Sexual behavior of female rats consists of motorically active solicitation behaviors and lordosis, the immobile reflexive posture of receptivity, which is elicited by dorsolateral flank and perivaginal somatosensory stimulation that is carried to the brainstem via the ventrolateral funiculi and integrated in the PAG (Pfaff and Modianos, 1985
To elucidate the possible role of the PAG in SK-induced kyphotic nursing, we used a functional marker of increased neuronal stimulation, activity of the immediate-early gene c-fos (Morgan and Curran, 1991
MATERIALS AND METHODS
Animals and housing. All methods received the prior approval of the Animal Care Committee of Rutgers University. Subjects were primiparous Long-Evans rats (Rattus norvegicus), and their litters were born and raised in our colony, as detailed by Stern and Lonstein (1996)
Fos protocol. To evaluate Fos-immunoreactivity, we studied 40 lactating females (n = 8 per group). Each dam was separated from her litter beginning at 7:30 A.M.-12:30 P.M. on day 5 for 48 hr before the test; we found previously that c-fos activation was unreliable after a 24 hr separation, whereas display of maternal behavior within a 1 hr test was unreliable after a 72 hr separation. At the time of separation, each dam was rehoused individually in a clean, clear, pan cage with fresh bedding and placed in a room that contained no dams with litters. Two hours before the test, dams were moved in their cage to the observation room to allow for habituation. Litters to be used as stimuli were placed for 44 hr with a foster lactating dam, after which they were placed in the incubator until the test 4 hr later. Fifteen minutes before the test, the mystacial pads of the pups were each injected subcutaneously with 0.025 ml of either isotonic saline (groups 1 and 3) or 2% lidocaine (group 2). We verified that by 15 min after injection of lidocaine (Xylocaine, Astra Pharmaceutical Products, Westborough, MA), mystacial pads were anesthetized completely (lack of response to a reasonably strong pinch with forceps) and that pups so treated were incapable of attaching to a nipple for ~75 min (Stern and Johnson, 1990
Lesion protocol. To evaluate the effects of cPAG lesions, we used 51 primiparous rats and their litters. In the first study (postpartum PAG-x), dams received bilateral sham (n = 10) or electrolytic (n = 13) lesions of the cPAG on day 7 postpartum. In the second study (prepartum PAG-x), subjects received bilateral sham (n = 11) or electrolytic (n = 17) lesions of the cPAG between days 15 and 17 of pregnancy. Between 8 and 9:30 A.M. beginning on day 1 (prepartum PAG-x) or day 5 (postpartum PAG-x), and continuing daily until the end of testing, each dam's rectal temperature was taken with a flexible rectal probe (Model 402) attached to a YSI telethermometer (Model 43TD; Yellowspring Instrument, Yellowspring, OH). Dams and litters were then weighed, and litters were cross-fostered between lesioned subjects, sham-operated controls, and surrogate dams.
Mother-litter interactions were observed continuously after a 3 hr dam-litter separation for 60 min on day 1-6 (prepartum PAG-x study) or for 45 min on day 6 (presurgically) and days 8-13 (postpartum PAG-x study). Maternal aggression was tested ~1 hr after the morning weighing by placing an unfamiliar male into the home cage opposite the nest. In the postpartum PAG-x study, the 10 min tests were carried out with the litter present on days 5 (presurgically), 8, and 10. Because attacks were frequent and pups were injured occasionally, in the prepartum PAG-x study the tests on days 2 and 6 occurred after removal of the litter and were reduced to 5 min.
Behavioral observations. A computerized event recorder (S & K Computer Products, Toronto, Ontario, Canada) was used for continuous observations, providing data on latency, frequency, and duration. Active behaviors directed toward pups included sniffing and licking, retrieval to the nest site, and mouthing (short-distance repositioning of the pups within the nest). Other active behaviors were self-grooming, exploration in or away from the nest (rearing, walking), digging/nesting (manipulation of bedding or paper), and eating or drinking.
Nursing behavior included hovering over the pups in the nest, while the dam was still motorically active, and motorically inactive kyphosis, with a low or high dorsal arch (Stern and Johnson, 1990 2 min of quiescence, not interrupted by >5 sec of activity. Recorded pup behaviors included active rooting on the dam's ventrum by at least one pup and stretching of the litter in response to milk receipt (Drewett et al., 1974
Aggressive behaviors of the dam (Stern and Kolunie, 1993
In the c-fos study, additional behaviors recorded for dams presented with the wire box, with or without pups inside, included sniffing, gnawing, or biting the box and standing on the box with at least two paws. NO STIM dams were observed briefly once every min for the first 10 min after cage top removal and then at 5 min intervals for the next 50 min; therefore, only calculated estimations of general activity are reported. If a dam was seen to display a particular behavior, she was assigned that behavior for the duration of the 1 or 5 min time block until the beginning of the next spot check.
In the lesion studies, nest construction was evaluated by providing dams with two double-ply 16 × 25 cm paper towels shredded and scattered in all quadrants of their home cage on the afternoons of days 5 and 9. Nests were rated early the next morning on a five-point scale (Numan and Callahan, 1980
Lesion surgery. Dams were anesthetized intraperitoneally with 70 mg/kg ketamine (Ketaset; Fort Dodge Laboratories, Fort Dodge, IA) and 5 mg/kg xylazine (Rompun; Bayer, Shawnee Mission, KS) and then placed in a Kopf stereotaxic instrument. Dams received bilateral sham or electrolytic lesions of the cPAG using the following coordinates: anteroposterior 7.4 mm from bregma, mediolateral ± 0.7 mm, and dorsoventral
Preparation of brain sections. Dams were anesthetized deeply with 150 mg/kg sodium pentobarbital (Nembutal; Abbott Laboratories, Chicago, IL). In the c-fos study, dams were then perfused through the heart with 200 ml of PBS, followed by 200 ml of 4% paraformaldehyde. Brains were removed, post-fixed for ~18 hr and placed in 18% sucrose/paraformaldehyde solution with 0.001% thimerosal at 4°C until they were sectioned within 7 d. Whole brains were cut into 50 µ coronal sections on a freezing microtome, and the sections were stored at 4°C in PBS, pH 7.4, until they were processed within 48 hr.
In the lesion studies, anesthetized dams were perfused though the heart with 150 ml of isotonic saline, followed by 150 ml of 10% formalin. Brains were removed and post-fixed for 24 hr, followed by submersion in 30% sucrose/formalin until they were sectioned. Brains were notched to maintain orientation, and the entire lesioned area was cut into 50 µ sections on a freezing microtome, mounted onto gelatinized slides, and dehydrated. Sections were then stained with potassium ferrocyanide followed by counterstaining with Neutral Red, cleared in xylenes, and coverslipped. The extent of each lesion, including missing and coagulated tissue plus the surrounding halo of damaged cells, was reproduced on sequential atlas figures (Paxinos and Watson, 1986
Immunocytochemistry was performed eight times; to reduce between- groups variability in background staining, sections from one subject per group were processed simultaneously, every fifth section throughout the entire brain being processed for each subject. After three 5 min rinses in PBS, sections were washed in 0.1 M glycine for 30 min, rinsed again three times, and washed in 0.5% hydrogen peroxide for 10 min. After they were rinsed in PBS, sections were incubated for 72 hr at 4°C with a rabbit affinity-purified polyclonal primary antibody (Oncogene Science, Manhassat, NY; lot 21930102; final dilution 1:750 in 0.3% Triton X-100 solution) raised against the peptide corresponding to residues 4-17 of human Fos. This antiserum reacts with Fos protein, Fos-related antigens, and Fos-Jun heterodimers (Oncogene Science). Sections were rinsed three times in PBS and incubated for 1 hr at room temperature (~22°C) with a biotin-SP-conjugated donkey anti-rabbit IgG secondary antibody (1:2500 in 3% normal donkey serum; Jackson ImmunoResearch Laboratories, West Grove, PA). After sections were rinsed, they were processed further with avidin-biotin-horse radish peroxidase complex (Vector Labs, Burlingame, CA), and the peroxidase enzyme was visualized with a 3-3 diaminobenzidine (0.0125%) solution containing 0.06% hydrogen peroxide and 0.015% NiCl. As a control procedure, the primary Fos antiserum was omitted on certain sections, which abolished Fos-immunoreactivity staining. Sections were mounted on gelatin-coated slides, dehydrated, and coverslipped. Another series of alternate sections for each subject was stained with cresyl violet to facilitate accurate location of neuroanatomical regions.
Quantification of cells with Fos-immunoreactive (ir) nuclei. One section per subject for each site was used for analysis and was chosen primarily for its correspondence to the reference atlas plate and secondarily for histological quality. No attempt was made to choose sections with the most Fos-ir labeling. Subjects were randomized and coded for analysis. Areas of investigation were viewed with a Nikon Optiphot 2 light microscope at a magnification of 125× using methods similar to those of Heeb and Yahr (1996) 
Fig. 1. Diagrammatic representation (from Paxinos and Watson, 1986
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All immunoreactive nuclei, regardless of intensity, lying fully within a particular square on the grid or overlapping its top or right edges (but not its bottom or left-hand borders), were directly quantified. This procedure eliminated the possibility of requantifying immunoreactive nuclei that lay on the shared border of two adjacent grid squares. All sites were analyzed by one observer (J.S.L.). The entire study (Lonstein et al., 1995
Data analyses. For the c-fos study, maternal and pup behaviors of the groups observed continuously were analyzed with the t-test or one-way ANOVA with Fisher's post hoc least squares difference (PLSD) test. Immunocytochemical results were analyzed with one-way ANOVAs and Fisher's PLSD tests. One subject in the BOX group was determined to be a statistical outlier in the number of Fos-ir cells analyzed (Dixon outlier test; p < 0.05). Sections from a subject in the P/B group were not analyzable because of inadequate perfusion and poor histology. These two subjects were replaced before completion of the study, and their brain sections were processed with those of five other subjects in the eighth immunocytochemical assay. For the lesion studies, behavioral data were analyzed with repeated measures ANOVA followed by Fisher's t post hoc tests. Nest construction data were analyzed with Mann-Whitney U tests. In the prepartum lesion experiment, one lesioned dam was an outlier with respect to the number of pups retrieved; these data were removed from the ANOVA.
RESULTS
Effects of maternal and nonmaternal stimuli on behavior and activation of c-Fos in the brain
Behavioral differences between groups Dams separated from their litters were highly maternal when reunited with pups 48 hr later, completing retrieval on average in <50 sec. Figure 2A shows that dams in all four groups with a stimulus in their cage spent the majority of their time either interacting with the stimulus or engaging in other activity. Dams with a box, however, showed less time in physical contact with their stimulus item than did dams with accessible pups. The sum of all active behaviors reveals that NSK dams were significantly more active than SK dams, which became quiescent while nursing (~34 vs ~23 min), but less active than dams with a box, either with pups inside or empty (~42-44 min) (F(3,28) = 7.23; p < 0.001). In contrast, all NO STIM dams were active in the first 10 min after brief removal of their cage tops, and then sporadically thereafter.
Fig. 2. Effect of different stimulus conditions on the behavior (mean ± SEM) of lactating dams during a 1 hr period. A, Stimulus-oriented perioral activity toward pups includes sniffing, licking, mouthing, and carrying of pups, and toward the box includes sniffing, gnawing, or biting the box; general perioral activity includes self-grooming, feeding, drinking, and tail retrieval; and general activity includes nest building, digging, and exploration (walking and rearing). The behavior of the No stimulus dams was estimated (see text). Statistically significant comparisons are indicated by different letters above the bars (p0.05). B, Hovering over (while active) and kyphotic crouching (while quiescent) by lactating dams physically interacting with SK or NSK pups. *, p
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Site differences in Fos-ir The only site at which SK dams had significantly more Fos-ir nuclei than NSK dams, which in turn had higher Fos-ir levels than the other groups, was the cPAG (Fig. 3). Labeling was found throughout the cPAG but tended to be denser in the lateral and ventrolateral regions (Fig. 4A-C). When subdivided into approximately equal thirds dorsoventrally (Table 1), similar group differences in Fos-ir labeling were found in the cPAG within the thirds ventrolateral and especially lateral to the cerebral aqueduct, whereas in the dorsolateral third, levels of Fos-ir were lower overall, and the pattern found in the other thirds was much less distinct. The three NSK dams showing short periods of kyphosis had levels of Fos-ir close to the mean of their group.
SK and NSK dams spent a comparable amount of time in the nest with their litter (Fig. 2B); however, NSK dams hovered over the litter while they were still active, including licking pups and self-grooming, twice as long as SK dams (F = 
Fig. 3. Number (mean ± SE) of Fos-ir nuclei in the rostral and cPAG expressed relative to the mean of the No Stimulus control group; statistically significant comparisons are indicated by different letters above the bars (p
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Fig. 4. Photomicrographs of cPAG in a representative dam in each of three groups: A, SK; B, NSK; C, no stimulus. D, Photomicrograph of rPAG in a representative NSK dam, where Fos is concentrated in the dorsal and dorsolateral regions. Scale bar, 100 µm.
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Table 1. Fos-ir nuclei (mean ± SE) in sites depicted in Figure 1 (n = 8/group)
Site SK NSK P/B BOX NO STIM F(4,35) p cPAG 199 ± 27a 128 ± 25b 60 ± 15c 49 ± 9c 22 ± 5c 15.80 0.0001 cPAGl 71 ± 7a 39 ± 11b* 15 ± 4c 12 ± 3c 4 ± 1c 18.76 0.0001 cPAGvl 91 ± 13a 59 ± 11b+ 28 ± 10c 17 ± 4c 11 ± 3c 14.01 0.0001 cPAGdl 37 ± 8a 30 ± 5ab 17 ± 5bc 20 ± 3bc 7 ± 2c 5.20 0.0021 rPAG 149 ± 22ab 236 ± 54a 97 ± 19b 124 ± 29b 81 ± 29b 3.71 0.0127 rPAGdl 50 ± 6ab 72 ± 15a 29 ± 6b 37 ± 8b 28 ± 6b 4.22 0.0068 rPAGdm 20 ± 5 18 ± 6 19 ± 5 22 ± 6 23 ± 6 0.12 0.9729 rPAGbal 79 ± 17ab 146 ± 43a 49 ± 13b 65 ± 18b 30 ± 11b 3.52 0.0161 PP 29 ± 4 26 ± 7 25 ± 6 33 ± 5 22 ± 7 0.42 0.7946 DR 39 ± 9 36 ± 10 17 ± 4 26 ± 5 16 ± 6 2.19 0.0910 Pn 314 ± 36 255 ± 52 272 ± 58 331 ± 31 178 ± 49 1.66 0.1810 SK, Suckled; NSK, nonsuckled; P/B, pups in wire-mesh box; BOX, empty wire-mesh box; NO STIM, no stimulus in cage during the 1 hr test period; c, caudal; r, rostral; l, lateral; vl, ventrolateral; dl, dorsolateral; dm, dorsomedial; bal, balance of rPAG. Significant differences between groups indicated by different superscript letters. SK > NSK; b+, p
In the rPAG, NSK dams had the highest levels of Fos-ir, significantly greater than the similarly low levels in P/B, BOX, and NO STIM dams, with intermediate levels seen in SK dams (Fig. 3). Labeling was confined primarily to the dorsomedial and dorsolateral aspects (Fig. 4D; Table 1). In fact, 46% of the total Fos-ir was found in the delineated dorsomedial and dorsolateral sections, which together represent only ~20% of the total area analyzed for the rPAG. No group differences in Fos-ir were found in the dorsomedial portion, but group differences similar to those observed for the entire rPAG were found in the dorsolateral portion and in the balance of rPAG. There were no significant group differences in Fos-ir labeling in the PP, DR, or Pn nuclei (Table 1). In addition, the ventral tegmental area and the spinal trigeminal nuclei (caudalis, interpolaris, oralis, principle) were found by inspection to have little or no Fos in any subject and therefore were not quantified. Effects of lesions on the cPAG
Histological analysis of prepartum PAG lesions Seven subjects with misplaced lesions were excluded from statistical analyses, four with only a unilateral lesion of the cPAGl,vl, one with a unilateral cPAGl,vl lesion and a contralateral superior colliculus lesion, one with a bilateral lesion of the dorsal cPAG and deep layers of the superior colliculus, and one with a bilateral lesion of the cPAG caudal to the intended site, which extended into the dorsal medulla and cerebellum. The remaining subjects (n = 10) all received bilateral damage to the cPAGl,vl, primarily at intercollicular levels, all lesions encompassing at least a 1.5 mm area from
Fig. 5. Reconstruction of representative prepartum (left) and postpartum (right) electrolytic lesions of the cPAGl,vl (modified from Paxinos and Watson, 1986
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Histological analysis of postpartum PAG lesions One dam with no discernible lesion and another with a misplaced lesion
more caudally in the ventrolateral PAG, bilaterally, with extension into the cerebellum and dorsomedial medulla
Motorically active behaviors PAG-x and control dams were remarkably similar in their display of active behaviors, whether the surgery was carried out pre- or postpartum (Table 2). Retrieval behavior was essentially normal after the cPAGl,vl was lesioned. Lesioned and sham groups were similar in the number of pups retrieved (Fig. 6A,B) and latency to retrieve the first pup (Fig. 6C,D). In the postpartum PAG-x study, both groups of dams retrieved fewer pups as the pups became older and more mobile (day F(1,6) = 10.4; p < 0.0001). Both pre- and postpartum PAG-x dams, however, tended to be slower to complete retrieval of each pup (Fig. 6E,F), mostly because of a delay of a few seconds between bringing the pup to the nest and actually depositing it there. Duration of licking (Fig. 6G,H), sniffing, and mouthing the pups did not differ between groups in either experiment. Time spent manipulating nest material and wood shavings was similar for both groups in the prepartum lesion experiment, but lower in postpartum PAG-x dams; however, ratings of nest construction of the postpartum dams on days 5 and 9 did not differ between groups (p > 0.05). Durations of other active behaviors, including self-grooming and cage exploration, were also not affected by cPAGl,vl lesions performed either pre- or postpartum (Table 2).
Table 2. Effects of lesions to the cPAGl,vl (PAG-x) carried out prepartum (gestation day 15-17) or postpartum (day 7) on rat dams' weight, body temperature, and behavior (means ± SEM) during a 60 or 45 min (respectively) interaction with pups
Measure Prepartum experiment: mean of days 1-6 Postpartum experiment: mean of days 8-13 Sham PAG-x F(1,19) Sham PAG-x F(1,19) Body weight (gm) 341 ± 8 350 ± 8 0.59 337 ± 6 334 ± 5 0.13 Body temperature (°C) 38.5 ± 0.1 38.6 ± 0.8 0.14 38.6 ± 0.1 38.6 ± 0.1 0.001 Latency (sec) Hover over 87 ± 3 149 ± 42 2.48 79 ± 8 107 ± 6 8.9* Quiescence 682 ± 72 1416 ± 84 44.1** 461 ± 44 1013 ± 61 50.6** Behavior duration (sec) Mouth pups 6.0 ± 1.0 5.8 ± 1.0 0.02 5.3 ± 1.1 9.9 ± 1.8 5.72* Nest, burrow 108 ± 22 67 ± 10 2.67 111 ± 28 41 ± 11 5.53* Self-groom 182 ± 18 181 ± 20 0.04 95 ± 27 93 ± 11 0.01 Walk, rear 160 ± 17 184 ± 37 0.38 108 ± 22 66 ± 12 2.22 Hover over 1283 ± 75 1536 ± 101 4.15 908 ± 79 934 ± 64 0.05 All nursing 1868 ± 102 1588 ± 107 3.58 1409 ± 129 1461 ± 85 0.08 Over + nursing 3151 ± 70 3124 ± 67 0.07 2317 ± 79 2396 ± 39 0.64 Partial kyphosis 96 ± 31 265 ± 76 4.61* 3.8 ± 2.9 11.6 ± 5.5 1.34 ANOVA values of data from postpartum experiment include the presurgery baseline data (day 6). p < 0.10; * p
Fig. 6. Effects of prepartum (left) or postpartum (right) sham (unfilled squares) or cPAG (filled circles) lesions on active maternal behavior (mean ± SEM) in daily 60 min (left) or 45 min (right) interactions with the litter, after a 3 hr dam-litter separation during the first (left) or second (right) week postpartum. A, B, Number of pups retrieved; C, D, latency (sec) to retrieve the first pup; E, F, (latency to retrieve the last pup)
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Nursing behavior Total time in physical contact with the pups during the daily observations did not differ between lesioned and sham groups operated on either pre- or postpartum (Table 2). Prepartum PAG-x dams spent ~15% more time actively hovering over their pups in the nest (F(1,19) = 4.2; p < 0.06), but postpartum PAG-x dams were similar to their controls (F(1,19) = 0.05; p > 0.8). Although the latency to begin quiescently nursing the pups from the beginning of the test was approximately twice as long for PAG-x dams compared with controls in both experiments, the total duration of quiescent nursing in all postures, ~50% of the observation time, did not differ significantly between groups on any day.
Once quiescent, however, PAG-x dams did not show normal nursing behavior. In prepartum PAG-x dams, kyphosis was reduced 85%, the full effect being present on the first test day, 7-9 d postsurgery (Fig. 7A). In postpartum PAG-x dams, kyphosis was similar in duration between groups presurgically on day 6 and was nearly eliminated after lesioning but not sham surgery, with no evidence of recovery; however, it took until 2 d after surgery (day 9) for the full effects of the lesion to be evident (Fig. 7B). For both experiments, the maximum sustained duration of kyphosis within a nursing bout was only 1-2 min in PAG-x dams versus 10-15 min in controls (Fig. 7C,D). In compensation for the near-absence of kyphosis, PAG-x dams nursed their litters primarily in the prone (Fig. 7E,F) and supine (Fig. 7G,H) positions during the entire postlesion period, uncharacteristic of controls in this testing situation. As a group, the seven prepartum lesioned dams with unilateral cPAG or misplaced lesions, or both, showed ~15 min of kyphosis each day of testing, which was 40% less than that displayed by sham controls but almost four times more than that shown by bilaterally cPAGl,vl-lesioned dams. 
Fig. 7. Effects of prepartum (left) or postpartum (right) sham (unfilled squares) or cPAG lesions on duration of nursing postures (mean min ± SEM) in daily 60 min (left) or 45 min (right) interactions with the litter, after a 3 hr dam-litter separation during the first (left) or second (right) week postpartum. A, B, Kyphosis; C, D, maximum sustained kyphosis within a nursing bout; E, F, nursing while lying prone over the litter; and G, H, nursing while supine on back or side. * p
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Litter weight gains All dams lactated postsurgery, 24 hr RLWGs being ~26% and ~15% less for litters interacting with prepartum and postpartum lesioned dams, respectively, compared with controls (F(1,19) = 18.2, p < 0.0004; F(1,19) = 7.1, p < 0.0155) (Fig. 8A,B). Litters of both lesioned and sham-lesioned dams gained weight during the daily 60 or 45 min dam-litter interactions, increasingly over time postpartum (p < 0.0001) (Fig. 8C,D); these gains were 16 and 20% less overall in litters suckling prepartum lesioned (F(1,19) = 1.26; p < 0.2755) and postpartum lesioned (F(1,19) = 6.49; p < 0.0197) dams. Although large lesions of serotonergic perikarya in DR reduce hypothalamic serotonin necessary for suckling-induced prolactin release (Borofsky et al., 1983
Fig. 8. Effects of prepartum (left) or postpartum (right) sham (unfilled squares) or cPAG (filled circles) lesions on indices of lactation (mean ± SEM) during the first (left) or second (right) week of lactation. A, B, Daily relative litter weight gains (gm); C, D, relative litter weight gains during daily 60 min (left) or 45 min (right) behavioral observations; and E, latency to first PSR, indicative of the dam's milk ejection. * p
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Maternal aggression On each test, all dams attacked the male at least once, but PAG-x dams were substantially more aggressive than controls (Table 3). Latency to attack from the beginning of the test was somewhat shorter in prepartum PAG-x rats (F(1,19) = 3.02; p < 0.10), especially on day 2, and in postpartum PAG-x rats only on day 10 (t(19) = 1.99; p = 0.06) (Fig. 9A,B). In the prepartum study, PAG-x dams displayed a significant increase in attack frequency compared with controls on both test days (1.7-fold in 5 min), whereas in the postpartum study, a significant postsurgical increase was evident on day 10 (2.4-fold in 10 min; Table 3). Similar differences were found in attack rate per minute calculated from the onset of attacking (prepartum: F(1,19) = 20.51, p < 0.001; postpartum: group × day, F(1,2) = 8.90, p < 0.001) (Fig. 9C,D). The mean attack duration per bout was 40% longer in prepartum PAG-x dams, but it did not differ between postpartum PAG-x and control dams (Table 3). In both pre- and postpartum experiments, the duration of aggressive grooming was significantly shorter after PAG-x (Table 3) and correlated significantly on the last test day with attack latency (r = 0.463 and 0.582, p < 0.04 and 0.01, respectively). Prepartum PAG-x dams displayed the sideways posture for half as long and boxing for more than twice as long as controls (Table 3). In both experiments, duration of sniffing did not differ between groups (Table 3), and there was little or no kicking. The seven prepartum PAG-x dams with unilateral cPAG or misplaced lesions, or both, showed aggressive behavior similar to that of sham controls (e.g., attack frequency means: 6 vs 7, day 2; 10 vs 8, day 6).
Pup stretch responses (PSRs), indicative of milk letdown, were reliably detectable from day 5 on; the number of PSRs did not differ between groups in either experiment. In the postpartum PAG-x study, PSRs during the daily mother-litter observation increased in frequency, from 4.3 to 7.1, day 6 to 13 (p < 0.0001), similarly for both groups. The latency to the first PSR from the beginning of the test was 38% longer on days 5 and 6 for prepartum PAG-x dams than sham controls (1599 ± 138 vs 997 ± 69 sec; F(1,19) = 16.2; p < 0.0007) and 26% longer across all postsurgical days in the postpartum experiment (F(1,19) = 10.59; p < 0.0042) (Fig. 8E). Latency of the last PSR did not differ between groups of postpartum lesioned dams, indicating more rapid milk letdowns in PAG-x dams after their late onset.
Table 3. Effects of lesions to the cPAGl,vl (PAG-x) carried out prepartum (gestation day 15-17) or postpartum (day 7) on lactating rat dams' behavior (means ± SEM) toward a strange male intruder (maternal aggression) in 5 or 10 min tests, respectively
Prepartum lesion: test day Postpartum lesion: test day 2 6 5 8 10 Measure/(F; p) Attack frequency (F = 18.94; p < 0.0003) (F = 9.2; p < 0.0005) Sham 6.5 ± 1.0 7.8 ± 0.7 14.0 ± 3.0 10.3 ± 1.8 7.9 ± 1.9 PAG-x 10.9 ± 1.4 13.0 ± 1.0 12.3 ± 2.3 13.8 ± 1.9 18.7 ± 1.7 Duration (sec) Sniff intruder (F = 0.74; ns) (F = 0.20; ns) Sham 105 ± 9 86 ± 8 123 ± 16 146 ± 15 138 ± 14 PAG-x 85 ± 8 89 ± 10 173 ± 15 151 ± 17 120 ± 12 Aggressive groom (F = 5.01; p < 0.04) (F = 8.35; p < 0.001) Sham 34 ± 6 30 ± 5 53 ± 10 78 ± 21 88 ± 21 PAG-x 21 ± 3 18 ± 6 96 ± 19 39 ± 12 48 ± 11 Attack bout (F = 15.12; p < 0.001) (F = 2.03; ns) Sham 3.6 ± 0.6 3.1 ± 0.4 5.5 ± 0.7 5.7 ± 0.7 5.0 ± 0.8 PAG-x 5.5 ± 0.5 5.7 ± 0.5 5.5 ± 0.7 6.5 ± 0.7 6.7 ± 0.6 Sideways posture (F = 2.14; ns) Sham 17 ± 9 31 ± 9 PAG-x 13 ± 4 10 ± 4 Boxing posture (F = 3.89; p < 0.10) Sham 6 ± 2 11 ± 4 PAG-x 29 ± 9 11 ± 5 Significant effects from ANOVA values based on Group (F(1.19)) in prepartum experiment and Group × Day interaction (F(1,2)) in postpartum experiment, which includes presurgery baseline (day 5); ns, no statistically significant effects. 
Fig. 9. Effects of prepartum (left) or postpartum (right) sham (unfilled squares) or cPAG (filled circles) lesions on aggressive behaviors (mean ± SEM) toward a strange male intruder during a 5 min (left) or 10 min (right) interaction. A, B, Latency to first attack (sec), and C, D, attack rate, calculated as the number of attacks per minute between the onset of attacking and the remainder of the test. * p
[View Larger Version of this Image (31K GIF file)]
DISCUSSION
Using visualization of Fos-ir, we identified a midbrain site, the lateral and ventrolateral regions of the PAG at intercollicular levels, that responds maximally to suckling stimulation, a pattern not found in six other brainstem structures, including the PAG rostral or caudal to this area (this report; Stern et al., 1997Effects of SK or NSK contact on Fos-ir expression in the PAG
SK stimulation specifically elevates c-fos compared with NSK stimulation in the cPAG, but not in >27 other brain sites, including the paraventricular and supraoptic nuclei (this report; Lonstein et al., 1995
NSK contact with pups resulted in significantly more Fos-ir in the cPAG than that occurring without physical interaction with pups. Recent studies revealed a contribution to this activation from trigeminal stimuli
NSK pup contact elicited the highest levels of Fos in the rPAG, especially dorsolaterally. This may reflect the effects of mPOA excitation during the performance of active maternal behavior (Numan, 1994 Comparison of pre- and postpartum PAG lesions
The effects of cPAG lesions on kyphosis and maternal aggression were apparent on the first postpartum tests, which occurred at least 6-7 d after prepartum lesioning, but required 2-3 d to be fully effective after the postpartum lesions. The direction of changes after postpartum lesioning suggests that previous experience with nursing or fighting did not ameliorate its effects. Rather, these differences are likely related to the time course of degenerative changes seen after traumatic brain injury. Significant degeneration of efferent or afferent connections of a lesioned region often requires at least 1 week (Gage et al., 1986
cPAG lesions performed either pre- or postpartum produced strikingly similar results, indicating effects on both the onset and maintenance of kyphosis and maternal aggression. In contrast, the integrity of the cPAG is not necessary for active maternal behaviors directed toward pups during either stage. Although the present results cannot definitively exclude a neuroendocrine basis for the reduced litter growth after cPAG lesions, the PAG is not in the known afferent path of suckling-induced prolactin and oxytocin secretion (Tindal, 1978 Role of the cPAG in kyphosis
Afferent fibers from the nipples project to widely overlapping regions of the spinal cord, primarily targeting dorsal horn laminae I and II (Tasker et al., 1986
Because suckling-induced nursing behavior in rats is necessarily accompanied by inhibition of motorically active behaviors such as retrieval and licking of pups (Voloschin and Tramezzani, 1979 Maternal aggression and the PAG
The integrity of the PAG is essential to the display of defensive behavior in cats and rats (Bandler and Depaulis, 1991
The PAG also modulates fear-motivated behavior (Fanselow, 1991
FOOTNOTES
Received Jan. 3, 1997; accepted Jan. 27, 1997.
This research was supported by National Institute of Mental Health Grant MH40459 (J.M.S.) and a Busch Biomedical Research Grant of Rutgers University (J.M.S., J.M. Swann). We thank Dr. Jennifer M. Swann and her laboratory assistants for their help in demonstrating the Fos immunocytochemistry procedures, Dr. Pauline Yahr and Ms. Michaela Heeb for their suggestions on Fos quantification, Ms. Danielle Simmons for assistance with behavioral observations, and Drs. M. David Egger and David Crockett and Ms. Sue Harris for help with the photomicrographs.
Correspondence should be addressed to Dr. Judith M. Stern, Department of Psychology, Rutgers University, New Brunswick, NJ 08903.
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ABSTRACT
