Several pilot experiments (35 animals) were conducted before the experimental protocols described above were finalized. Initially, SD was induced as previously described by Kraig et al. (1991)
. Briefly, 1 m
KCl was applied to the exposed parietal cortex of one cerebral hemisphere while 1 m
NaCl was applied to the contralateral side for a period of 30 min, 1 hr, or 2 hr. Furthermore, animals were killed by perfusion-fixation as described above immediately after the recording periods. Others were allowed to awaken for 24 hr before being reanesthetized and killed by perfusion–fixation. In still other experiments, the anesthetic was changed from halothane to pentobarbital (given as a 50 mg/kg intraperitoneal injection). None of these experimental paradigms were sufficiently sensitive to detect a change in left/right labeling caused by SD. We speculate that the effects of SD within TNC were obscured by the trauma associated with drilling of craniotomies, opening of the dura, and application of KCl to the skull and dura. Accordingly, the dura was left intact in our final preparation. In addition, KCl was not applied to the dura, but instead was microinjected 1000 μ
m below the pial surface via a silica needle that pierced the intact dura. Furthermore, animals were allowed to recover for 6−7 hr after surgery before being reanesthetized for the induction of recurrent SD.
Together, these technical innovations allowed us to show that recurrent SD significantly increases c-fos protein staining within the ipsilateral TNC. Based on our present knowledge of the employed antisera, we cannot say with certainty whether the observed immunoreactivity represents c-fos protein itself or a related antigen(s).
Physiologic variables were similar in all experimental groups (). The mean number of SDs for each experimental group are shown in . Continuous records representative of the treatment groups that evoked SDS are shown in . SD always occurred after microinjection of 1 m KCl [group II, 6.6 ± 1.0 SD (range, 6−8)] or 0.15 m KCl [group VI, 6.3 ± 1.0 SD (range, 5−7)]. Treatment with intravenous sumatriptan or chronic sensory denervation did not affect the number of SDS [group III, 6.2 ± 0.4 SD (range, 6−7); group IV, 7.0 ± 1.4 SD (range, 6−9)]. Sumatriptan pretreatment did, however, slightly alter the DC waveform of SD recorded in frontal cortex (). The waveform was sharper with less plateau. The significance of this alteration is unknown. Similarly, in the example shown, spontaneous electrical activity stopped at the onset of the fourth SD (evidenced by the narrowing of the baseline DC signal). Such a diminution in baseline activity was not regularly seen in the sumatriptan group. Combined hyperoxia and hypercapnia reduced the number of evoked SDS after KCl microinjection [group V, 4.4 ± 0.5 SD (range, 4−5); group VII, 2.5 ± 1.2 SD (range, 1−4)]. Microinjection of 1 m NaCl (group I) never evoked SD, although insertion of the injection cannula did evoke a single SD in one animal [0.2 ± 0.4 SD (range, 0−l)].
Number of spreading depressions and c-fos expression evoked in the cortex by microinjection of NaCl or KCl
Figure 1 Spreading depression (SD) frequency. The upper panel shows a continuous DC record from one animal that received 0.5 μl/min injection of 1 m KCl every 9 min for 1 min in left posterior parietal cortex. Arrows (pointing up) at the bottom indicate (more ...)
C-fos protein response in forebrain
C-fos protein–LI was expressed intensely within cerebral cortex on the side ipsilateral to SD (, ). Sumatriptan or chronic sensory denervation did not reduce the degree of neocortical staining induced by SD (, ). Positive cells were particularly evident in laminae II, III, and VI. Staining was reduced after combined hyperoxia and hypercapnia and was only faintly observed after NaCl microinjection ().
Figure 2 c-fos LI in the cingulate cortex taken from the frontal region after animals received microinjections into left posterior parietal cortex of 1 m NaCl (a), 1 m KCl (b), 1 m KCl/sumatriptan (c), 1 m KCl/sensory denervation (d), or 1 m KCl/hyperoxia-hypercapnia (more ...)
Positive cells were also noted within ipsilateral hippocampus in at least half the animals after KCl microinjections, all animals after KCl/sumatriptan treatment, and four out of five animals after KCl/sensory denervation. After NaCl microinjections, one of six animals did show prominent ipsilateral hippocampal staining; this animal exhibited a single SD during the recording period. Only faint c-fos protein staining was present in the KCl/hyperoxia-hypercapnia group.
Many labeled cells were observed bilaterally in the thalamus such as habenular, paraventricular thalamic, mediodorsal, intermediodorsal, and centromedian nuclei, in the hypothalamus such as supraoptic, paraventricular, and dorsomedial nuclei, in the medial, lateral, and triangular septal nuclei, and in the medial and lateral preoptic area. There were no apparent differences between groups, however. A few c-fos protein–labeled cells were observed in the medial and mediodorsal parts of caudate-putamen bilaterally after 1 m KCl injection, and to a lesser degree after 1 m NaCl injection, but c-fos protein expression was not reduced after KCl injection that accompanied combined hyperoxia and hypercapnia, or sumatriptan treatment or after chronic meningeal deafferentation. Rare c-fos protein–labeled cells were detected in globus pallidus.
C-fos protein response in brainstem
C-fos protein–labeled cells were observed bilaterally within multiple brainstem nuclei (). The numbers were greatest within TNC, parabrachial nucleus, nucleus of the solitary tract, dorsal raphe nucleus, periaqueductal gray, and inferior olive. The numbers of positive cells within nucleus of the solitary tract and dorsal raphe nucleus did not differ between groups (data not shown). The numbers of positive cells within parabrachial nucleus, periaqueductal gray, and inferior olive were greater in the KCl groups. However, only within parabrachial nucleus were fewer positive cells counted after 1 m KCl/sumatriptan, 1 m KCl/sensory denervation, or 1 m KCl/hyperoxia-hypercapnia treatment [146 ± 25 (1 m KCl), 100 ± 13 (1 m NaCl, p < 0.05), 69 ± 15 (1 m KCl/sumatriptan, p < 0.01), 74 ± 19 (1 m KCl/sensory denervation, p < 0.01), 73 ± 19 (1 m KCl/hyperoxia-hypercapnia, p < 0.01)].
Expression of c-fos protein in brainstem nuclei
C-fos protein response in TNC
Labeled cells were found throughout TNC bilaterally in all groups (, ). Positively stained cells were located chiefly in laminae I and IIo, and were particularly evident within the ventro-lateral segment corresponding to the region receiving trigeminal afferent inputs from the ophthalmic division (). Sparse labeling was present in laminae III–V.
Figure 3 Ratio of the number of c-fos–immunoreactive cells (left: right sides) at five different levels within TNC (laminae I and 110) after microinjections in left parietal cortex of 1 m NaCl (n = 6, open bars), 1 KCl (n = 7, solid bars), 1 m KCl/sumatriptan (more ...)
Figure 4 Immunoreactive c-fos–containing cells in TNC from sections (50 μm, −3.0 mm caudal to obex) on left (a, c, e, g, i) and right (b, d, f, h, j) sides from animals representative for each group after left parietal microinjections of (more ...)
Most positive cells were located between −1.5 and −4.5 mm from obex. Consequently, labeled cells were counted above and below −1.5 mm to define a rostral and caudal TNC part, respectively. Approximately 80% of total positive cells were found in the caudal segment [74 ± 15 (caudal) as compared to 45 ± 14 (rostral) cells per section after 1 m
KCl]. Differences between the experimental groups were more significant in the caudal segment as well. For the above reasons, and because the spinal portion of the TNC corresponds to the termination sites for the ophthalmic division (Darian-Smith, 1966
), the results obtained for rostral and caudal TNC will be described separately.
The greatest number of c-fos protein–positive cells was found after 1 m KCl microinjections alone (right plus left; 74 ± 15, p < 0.01). Labeled cells ipsilateral to the KCl microinjection accounted for this increase. The numbers for the other treatment groups are as follows: 61 ± 11 (1 m NaCl), 35 ± 11 (1 m KCl/sumatriptan), 47 ± 13 (1 m KCl/sensory denervation), and 21 ± 6 (1 m KCl/hyperoxia–hypercapnia). Fewer cells were counted after 0.15 m KCl [74 ± 15 (1 m KCl) vs. 25 ± 6 (0.15 m KCl)]. There were no significant differences between groups treated with 0.15 m KCl and 0.15 m KCl/hyperoxia-hypercapnia (25 ± 6 vs. 21 ± 9, respectively).
When the data are expressed as the ratio of positive cells on the two sides (left : right), variability between individual experiments (e.g., due to differences in chromogen development, etc.) is taken into consideration. When expressed in this way, the differences between groups were highly significant (p < 0.01). The ratios (as well as number of cells per section on the left vs. right sides, respectively) were 1.37 ± 0.19 (43 ± 10 vs. 31 ± 6, 1 m KCl) and 1.35 ± 0.08 (15 ± 4 vs. 11 ± 3, 0.15 m KCl), respectively. The groups treated with sumatriptan or sensory denervation did not show significant differences between the two sides [1.13 ± 0.11, p < 0.05 (18 ± 6 vs. 16 ± 5), 1 m KCl/sumatriptan; 1.11 ± 0.12, p < 0.05 (24 ± 6 vs. 23 ± 7), 1 m KCl/sensory denervation], nor did the animals microinjected with NaCl [1.06 ± 0.06, p < 0.01 (32 ± 6 vs. 30 ± 5)]. Similarly, combined hyperoxia and hypercapnia administered prior to 1 m KCl or 0.15 m KCl significantly reduced the ratio and differences in cell numbers between the two sides [1.09 ± 0.10, p < 0.01 (10 ± 2 vs. 10 ± 3), 1 m KCl; 1.04 ± 0.05, p < 0.01 (11 ± 5 vs. 10 ± 4), 0.15 m KCl)].
Group differences based on total cell numbers were not found in this segment of TNC.
Statistically significant differences were noted between the two sides, however, after 1 m KCl microinjection versus 1 m NaCl microinjection (1.17 ± 0.06, p < 0.05). The number of positive cells per section on the left and right sides was 25 ± 8 and 22 ± 7, respectively. No significant asymmetries occurred in those animals treated with 0.15 m KCl, 1 m NaCl, 1m KCl/sumatriptan, 1 m KCl/sensory denervation, or KCl/hyperoxia-hypercapnia, however.