NGFI-A expression in the rat brain after sleep deprivation

Molecular Brain Research 46 Ž1997. 143–153

Research report

NGFI-A expression in the rat brain after sleep deprivation Maria Pompeiano b

a,)

, Chiara Cirelli b, Simonetta Ronca-Testoni a , Giulio Tononi

b

a Istituto di Chimica Biologica, UniÕersita’ di Pisa, Õia Roma 55, I-56126 Pisa, Italy Dipartimento di Fisiologia e Biochimica, UniÕersita’ di Pisa, Õia S. Zeno 31, I-56127 Pisa, Italy

Accepted 22 October 1996

Abstract The effects of total sleep deprivation ŽSD. on the expression of the immediate-early gene NGFI-A were studied in the rat brain by in situ hybridization. Rats were manually sleep-deprived for 3, 6, 12 and 24 h starting at light onset Ž08:00 h. and for 12 h starting at dark onset Ž20:00 h.. SD performed during the day induced a marked increase in NGFI-A mRNA levels with respect to sleep controls in many cerebrocortical areas and caudate-putamen, which was most evident after 6 h SD. A decrease was seen in hippocampus and thalamus, particularly after 12 h SD. Rats sleep-deprived for 12 h during the night showed an increase in NGFI-A expression in some cortical areas while rats sleep-deprived for 24 h showed few changes with respect to controls. The pattern of NGFI-A expression after forced wakefulness showed some differences from that observed after spontaneous wakefulness wM. Pompeiano, C. Cirelli and G. Tononi, Immediate early genes in spontaneous wakefulness and sleep: expression of c-fos and NGFI-A mRNA and protein, J. Sleep Res., 3 Ž1994. 80–96x. These observations are discussed with respect to the functional consequences of wakefulness in specific brain areas. Keywords: Brain; Immediate-early gene; NGFI-A; Rat; Sleep; Sleep deprivation; Wakefulness; Zifr268

1. Introduction We have observed that the expression of two immediate-early genes ŽIEGs., c-fos and NGFI-A, shows notable changes in the rat brain with respect to spontaneous wakefulness and sleep w39x. Changes in c-fos expression were also seen after variable periods of sleep deprivation ŽSD. w8,13,32,37x. These findings indicate that the functional consequences associated with sleep and wakefulness are probably reflected in molecular changes in specific brain areas, as had been suggested by earlier studies w2,34,41x. In the present study, we investigated the effects of variable periods of SD on the expression of NGFI-A. NGFI-A, also known as zifr268, egr-1 or krox 24 and sometimes referred to by the acronym ZENK, is rapidly and transiently induced in the rat brain by several stimuli, such as sensory stimulation w56x and experimental seizures Že.g. w43x.. NGFI-A expression has been used, like c-fos expression, as a marker of neural activity w43,56,58x. Since NGFI-A protein acts as transcription factor regulating the

) Corresponding author. Istituto di Chimica Biologica, Scuola Medica, via Roma 55, I-56126 Pisa, Italy. Fax: q39 Ž50. 55-0241; E-mail: [email protected]

expression of target genes Že.g. w33x., NGFI-A can be considered as a potential marker of genetic activation. One of the reasons to investigate this IEG in addition to c-fos is that some neuronal populations may constitutively be unable to express one or the other IEG w14,18x. Moreover, NGFI-A shows some differences from c-fos and displays some peculiar characteristics that make it an interesting candidate to be examined. While c-fos has low basal levels of expression w19x, NGFI-A is constitutively expressed at high levels in several brain areas w44x. This facilitates the evaluation of reductions as well as of increases in NGFI-A expression. NGFI-A seems to be involved in the normal processing of sensory information w44,58x and its induction seems to reflect changes in physiological activation more rapidly than c-fos w57x. There is also strong evidence that NGFI-A may be involved in the production of long-term synaptic changes, such as those occurring during LTP and other learning-related phenomena w17,42x. The visualization of IEG expression levels represents a tool to map ‘activated’ brain areas which could subserve specific tasks in relation to sleep regulation and function. Changes in the levels of transcription factors associated with different arousal states may have important functional consequences on the expression of many target genes. In addition, the comparison of patterns of IEG expression obtained after spontaneous and forced wakefulness may

0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 6 . 0 0 2 9 5 - 1

Cerebral cortex: Frontal Motor Parietal Temporal Occipital Cingulate Retrosplenial Insular Perirhinal Piriform Entorhinal Olf bulb Anterior olf N Dorsal endopiriform N Basal gangliar rel areas: Caudate-putamen Accumbens N Ventral pallidum Globus pallidus Bed N stria terminalis Basolat-central amygd NN Lateral olf tract N Claustrum Hippocampusr septum: CA1 CA2–3 Dentate gyrus Presubiculum Lateral septal N Medial septal N Septohypothalamic N Hypothalamus: Anterior hypoth area Medial preoptic area Lateral preoptic area Suprachiasmatic N Supraoptic N Dorsomedial N Ventromedial N

Region 2.54"0.09 a 3.89"0.20 3.34"0.31 4.16"0.16 4.39"0.26 a 2.87"0.13 a 2.79"0.10 1.53"0.09 2.86"0.08 a 2.23"0.23 2.48"0.15 19.34"0.51 16.06"1.32 1.66"0.08 1.97"0.11 1.26"0.04 0.89"0.02 1.13"0.03 1.21"0.05 2.15"0.06 2.42"0.32 1.89"0.09 2.90"0.06 2.30"0.04 2.42"0.05 a 3.11"0.19 1.35"0.02 1.14"0.05 1.08"0.03 1.26"0.04 1.05"0.03 0.99"0.02 1.45"0.06 1.26"0.04 1.45"0.05 1.49"0.06

1.72"0.02 1.40"0.05 0.97"0.02 1.22"0.04 1.26"0.02 1.93"0.10 1.61"0.14 1.97"0.12

3.33"0.12 2.54"0.07 3.11"0.13 2.89"0.17 1.48"0.04 1.32"0.07 1.06"0.03

1.43"0.09 1.06"0.03 1.06"0.03 1.51"0.05 1.25"0.05 1.38"0.05 1.42"0.07

3 hL SD

1.85"0.09 3.12"0.19 2.70"0.13 3.43"0.22 3.17"0.13 2.19"0.08 2.62"0.12 1.66"0.10 2.22"0.14 2.19"0.10 2.08"0.12 18.33"1.43 13.15"0.87 1.68"0.08

3 hL C

1.35"0.06 1.37"0.03 1.16"0.02 1.29"0.12 1.26"0.08 1.71"0.03 1.59"0.03

3.40"0.12 2.73"0.13 3.29"0.09 3.55"0.14 1.47"0.03 1.17"0.05 1.15"0.03

1.59"0.04 1.27"0.05 0.97"0.02 1.06"0.04 1.34"0.04 2.23"0.12 2.02"0.14 1.79"0.06

1.97"0.05 3.68"0.09 3.43"0.16 3.24"0.14 2.92"0.18 1.78"0.05 3.08"0.05 1.63"0.03 2.87"0.12 2.25"0.17 2.15"0.06 17.77"0.76 12.14"0.47 1.52"0.05

6 hL C

Table 1 NGFI-A mRNA levels in sleep-deprived ŽSD. and respective control rats ŽC.

1 .39"0.04 1.49"0.11 1.23"0.08 1.46"0.07 1.31"0.08 1.65"0.08 1.61"0.08

3.01"0.06 2.23"0.06 2.73"0.11 a 3.72"0.20 1.51"0.03 1.19"0.05 1.18"0.08

a

1.38"0.04 1.08"0.06 1.06"0.07 1.39"0.05 1.52"0.09 1.48"0.04 1.47"0.04

2.94"0.07 2.76"0.05 2.72"0.05 3.20"0.08 1.65"0.03 1.51"0.07 1.10"0.02

1.92"0.05 1.77"0.05 0.98"0.05 1.27"0.04 1.29"0.04 1.89"0.06 2.09"0.09 2.13"0.05

a

2.18"0.08 1.45"0.06 1.06"0.03 1.05"0.02 1.39"0.06 2.77"0.06 1.81"0.14 2.54"0.10

2.26"0.11 2.63"0.05 2.77"0.03 3.05"0.10 3.42"0.09 2.23"0.05 2.43"0.03 2.09"0.06 2.36"0.08 2.45"0.05 2.22"0.22 13.74"0.25 10.18"0.31 1.89"0.05

12 hL C

2.93"0.09 a 4.45"0.19 4.67"0.21 a 4.65"0.16 a 4.66"0.18 a 2.22"0.05 a 4.26"0.22 a 2.38"0.10 a 3.37"0.10 3.28"0.26 2.16"0.10 16.65"0.21 12.65"0.26 2.16"0.08 a

6 hL SD

a

1.20"0.02 1.04"0.04 1.11"0.02 1.19"0.03 1.22"0.03 1.37"0.08 1.36"0.10

2.40"0.11 2.26"0.11 2.17"0.08 2.85"0.16 1.47"0.04 1.24"0.07 1.14"0.03

2.54"0.03 a 2.36"0.06 a 2.15"0.04 a 3.23"0.08 1.57"0.02 1.49"0.06 1.04"0.03 1.37"0.03 1.02"0.03 0.95"0.03 1.52"0.05 1.50"0.08 1.28"0.02 1.29"0.03

2.39"0.09 1.68"0.15 1.01"0.03 1.06"0.03 1.17"0.02 2.62"0.11 2.62"0.15 2.55"0.08

3.02"0.09 3.65"0.15 3.93"0.10 3.27"0.22 4.86"0.25 2.28"0.10 3.07"0.04 2.47"0.07 3.48"0.18 2.37"0.15 2.30"0.10 21.54"0.62 17.87"0.51 2.10"0.08

12 hDr24 h C

2.11"0.02 1.76"0.03 0.98"0.03 1.28"0.06 1.17"0.02 2.27"0.08 2.80"0.20 2.31"0.04

2.76"0.04 a 2.70"0.06 2.50"0.03 a 3.61"0.08 a 3.84"0.10 2.43"0.05 2.23"0.06 2.18"0.05 3.01"0.06 a 2.68"0.05 2.50"0.09 11.37"0.29 a 10.23"0.17 2.12"0.03 a

12 hL SD

1.13"0.04 1.00"0.03 1.09"0.03 1.22"0.06 1.16"0.06 1.21"0.04 1.27"0.05

2.57"0.10 2.03"0.06 1.79"0.07 3.05"0.16 1.44"0.04 1.34"0.07 1.11"0.05

2.67"0.10 1.89"0.10 0.99"0.04 1.03"0.04 1.13"0.03 2.82"0.23 2.83"0.18 2.56"0.13

4.14"0.18 a 4.35"0.25 4.90"0.20 4.94"0.23 a 6.06"0.29 3.12"0.19 2.83"0.13 2.52"0.12 3.47"0.18 3.06"0.21 2.56"0.11 22.60"1.09 21.75"0.56 a 2.12"0.11

12 hD SD

1.26"0.06 1.07"0.07 1.06"0.03 1.37"0.08 1.21"0.05 1.29"0.05 1.25"0.03

2.50"0.05 2.01"0.07 1.80"0.08 2.29"0.15 1.48"0.04 1.31"0.07 1.10"0.05

2.64"0.09 1.86"0.09 0.97"0.02 1.06"0.03 1.20"0.02 3.02"0.17 2.69"0.28 2.69"0.28

3.12"0.09 b 3.25"0.06 4.72"0.29 3.76"0.39 4.91"0.25 2.55"0.10 3.40"0.14 2.52"0.10 3.59"0.21 2.75"0.13 2.50"0.22 24.64"1.05 24.82"1.01 a 2.06"0.09

24 h SD

144 M. Pompeiano et al.r Molecular Brain Research 46 (1997) 143–153

1.36"0.14 1.37"0.04 1.89"0.05 1.44"0.03 1.50"0.08 1.50"0.04 1.33"0.07 1.25"0.02 1.37"0.02 1.77"0.05 1.49"0.05 1.87"0.08 1.67"0.04 1.55"0.06 1.58"0.06 1.20"0.06 1.49"0.03 1.45"0.06 1.35"0.05 1.24"0.04 1.60"0.10 1.20"0.02 2.97"0.14

1.58"0.05 1.35"0.10 2.19"0.09 1.61"0.08 1.49"0.09 1.62"0.05 1.33"0.07 1.45"0.03 1.43"0.04 1.60"0.06 1.40"0.06

1.76"0.05 1.73"0.09 1.55"0.05 1.47"0.05 1.25"0.06 1.57"0.06 1.36"0.06 1.34"0.02 1.32"0.01 1.59"0.09 1.31"0.03 3.15"0.13

a

1.84"0.09 1.71"0.07 1.98"0.10 1.35"0.04 1.30"0.03 1.97"0.10 1.53"0.07 1.28"0.03 1.35"0.03 1.79"0.08 1.23"0.05 3.00"0.10

1.72"0.05 1.65"0.09 2.34"0.06 1.86"0.04 2.03"0.04 1.95"0.03 1.91"0.04 1.86"0.04 1.61"0.02 1.95"0.09 1.67"0.04 1.78"0.08 1.81"0.10 1.62"0.08 1.33"0.05 1.35"0.04 2.14"0.09 1.69"0.10 1.31"0.02 1.29"0.03 1.76"0.09 1.09"0.03 2.82"0.09

1.47"0.04 a 1.51"0.05 1.96"0.11 1.53"0.10 2.03"0.15 1.70"0.11 1.73"0.15 1.49"0.09 1.49"0.07 1.72"0.07 1.61"0.06 2.07"0.04 1.97"0.06 1.70"0.08 1.55"0.04 1.37"0.05 1.71"0.06 1.80"0.11 1.21"0.01 1.30"0.03 1.77"0.06 1.36"0.03 3.63"0.13

1.44"0.03 1.39"0.04 2.06"0.04 1.49"0.03 1.73"0.06 1.54"0.03 1.63"0.07 1.45"0.02 1.59"0.02 1.68"0.06 1.12"0.01 1.89"0.07 1.95"0.07 1.56"0.04 1.49"0.04 1.56"0.08 1.79"0.03 1.41"0.06 1.33"0.04 1.36"0.05 1.59"0.06 1.44"0.07 3.18"0.13

1.36"0.03 1.45"0.05 1.55"0.02 a 1.20"0.01 a 1.19"0.02 a 1.15"0.01 a 1.08"0.01 a 1.15"0.02 a 1.34"0.02 a 1.58"0.03 1.10"0.01 2.68"0.07 2.28"0.09 1.31"0.03 1.30"0.03 1.49"0.11 1.68"0.05 1.65"0.06 1.24"0.07 1.33"0.07 1.59"0.07 1.22"0.02 3.16"0.19

1.67"0.05 1.57"0.08 1.48"0.06 1.14"0.04 1.24"0.05 1.17"0.02 1.12"0.05 1.23"0.04 1.38"0.06 1.56"0.03 1.27"0.03 2.35"0.11 2.16"0.12 1.28"0.06 1.32"0.03 1.38"0.05 1.64"0.09 1.48"0.06 1.18"0.02 1.23"0.02 1.50"0.04 1.21"0.03 3.48"0.14

1.53"0.07 1.49"0.10 1.40"0.06 1.11"0.03 1.19"0.05 1.11"0.03 1.07"0.05 1.13"0.03 1.33"0.05 1.66"0.05 1.20"0.04

2.21"0.10 2.11"0.10 1.28"0.06 1.20"0.05 1.35"0.03 1.77"0.10 1.22"0.03 a 1.41"0.08 1.30"0.03 1.59"0.07 1.29"0.02 3.34"0.15

1.58"0.06 1.61"0.14 1.32"0.07 1.05"0.02 1.22"0.09 1.12"0.03 1.09"0.06 1.08"0.03 1.20"0.05 1.50"0.05 1.20"0.03

The intensity of the hybridization signals was expressed as optical density ratio ŽODR.. ODRs are obtained by dividing the optical densities measured for each structure on X-ray films by the optical density of the white matter measured from the same section. For the olfactory bulbs and nucleus, the slide background signals were used. ODR are expressed as mean"SEM. For each region, ODRs were measured in two consecutive sections on both sides of the brain for each subject Ž3 subjectsrcondition.. Differences between measurements obtained from SD and the corresponding C rats were evaluated using the BonferronirDunn post-hoc test corrected for the appropriate number of multiple comparisons, with the P - 0.05 criterion level. a SD vs. respective C; b 24 h SD vs. 12 hL SD. amygd, amygdaloid; basolat, basolateral; hypoth, hypothalamic; N, nucleus; NN, nuclei; olf, olfactory; rel, related; ret, reticular; tegm, tegmental.

Thalamus: Anterior nuclear group Paraventricular N Medial habenular N Lateral habenular N Centromedial N Centrolateral N Rhomboid-reuniens NN Laterodorsal N Ventroposterior NN Medial geniculate N Zona incerta Brainstem: Superior colliculus Inferior colliculus Red N Central gray Dorsal raphe Pontine NN Subbrachial N Dorsal tegm N Laterodorsal tegm N Locus coeruleus Gigantocellular ret N Cerebellar cortex

M. Pompeiano et al.r Molecular Brain Research 46 (1997) 143–153 145

146

M. Pompeiano et al.r Molecular Brain Research 46 (1997) 143–153

point to functional differences between these two conditions at the molecular level. The effects of short periods of SD on NGFI-A mRNA levels were investigated in the rat brain by using in situ hybridization techniques. The SD periods examined were 3, 6, 12 and 24 h starting at 08:00 h and 12 h starting at 20:00 h. The shortest SD period, 3 h, was selected because it appears to be sufficient to trigger homeostatic mechanisms of sleep regulation Že.g. w12,51x. and it is compatible with the time constants of IEG expression. 12 h SD were performed both during the light and the dark hours, to reveal the interaction between the circadian and homeostatic components of sleep regulation w22,30,50x. 24 h SD was the longest SD period examined to avoid significant stress to the animal w52x and a possible plateau effect of sleep homeostatic mechanisms w25,35x. Part of this work was published in short form w38x. 2. Materials and methods 2.1. Animal recording and SD procedure 27 adult male WKY rats Ž300 " 50 g, Charles River, Calco, Italy. were individually housed and maintained in a controlled environment ŽLD 12:12, light on at 08:00 h; light intensity 100 candlesrm2 ; 23 " 18C.. The rats were implanted with silverrsilver chloride ball electrodes for chronic EEG recordings and silver electrodes were placed bilaterally in the neck mucles for EMG recordings Žsee w39x for details.. Frontal and hippocampal EEGs were recorded along with neck EMG during recovery from surgery and manually scored on paper, until the percentages and distribution of sleep states were within published norms w3x. Wakefulness was characterized by mixedfrequency, low-voltage frontal EEG in the presence of neck muscle tonus, NREM sleep Žnon-REM or slow-wave sleep. by low-frequency, high-voltage EEG and sleep spindles and REM sleep by mixed-frequency, low-voltage frontal EEG, regular u Ž5–8 Hz. rhythm in the hippocampal EEG and absence of neck muscle tonus. A tactile stimulation protocol was used for total SD, consisting in brief strokes Ž1–2 s. not restricted to a single part of the body. Total SD was performed for 3 h Ž3 hL SD, n s 3., 6 h Ž6 hL SD, n s 3. and 12 h Ž12 hL SD, n s 3. during the light period ŽL. starting at 08:00 h; for 12 h Ž12 hD SD, n s 3. during the dark period ŽD. starting at 20:00 h; and for 24 h Ž24 h SD, n s 3. starting at 08:00 h Žfor details, see w9x.. After SD, rats were anesthetized with ether in their cages and killed by decapitation. Control ŽC. rats exposed to the same environmental conditions were killed with the same procedure at the same phase of the 24-h cycle Ž3 hL C, 6 hL C and 12 hL C, n s 3 each; 12 hD and 24 h shared C, n s 3.. Brains were quickly removed, frozen on dry ice and stored at y708C until sectioned. Frontal sections Ž20 m m thick. of the entire brain were cut at the cryostat according to the atlas of

Paxinos and Watson w36x, thaw-mounted on gelatin-coated slides and kept at y208C until use. To minimize variability due to incubation procedures and to facilitate comparisons, sections of the same levels from a SD animal and a corresponding control were placed on the same slides. Sections from 12 hD and 24 h SD and the shared C were placed on the same slides. 2.2. In situ hybridization An antisense oligodeoxynucleotide was used complementary to the base-sequence coding for amino acids 1–16 of the NGFI-A protein w29x. It was labeled with w a32 x P dATP Ž3000 Cirmmol; DuPont NEN, Boston, MA. using terminal transferase ŽBoehringer, Mannheim.. The specificity of the hybridization signal was verified by a series of control experiments w39x. Tissue sections were fixed, hybridized overnight with the labelled probe and then washed as described w39x. Autoradiograms were generated by apposition to b-max film ŽAmersham, UK. for 10 days. The film autoradiographs were quantified by using a computer-assisted image analysis system ŽMCID, St. Catherine’s, Ontario.. The optical density measured for each region of each subject was divided by the optical density of the white matter measured from the same section, to obtain an optical density ratio ŽODR.. In the case of the olfactory bulbs and nuclei, the ratio was calculated using the slide background signal. For each region, ODRs were collected from two consecutive sections on both sides of the brain. Thus, each region on each side of the brain has two replicate measures. A nested ANOVA was performed over all brain regions with replicates, side Žright or left. and condition ŽC or SD. nested within region as factors. BonferronirDunn post-hoc tests Žcorrected for the appropriate number of multiple comparisons. were performed with the P - 0.05 criterion level to identify regions which changed with the experimental conditions. 3. Results 3.1. SD Control rats showed percentages and distribution of wakefulness, NREM and REM sleep in accordance with standard values w3x. In SD rats, REM sleep was always totally suppressed. NREM sleep was also suppressed, with the only exception of short periods at the end of the light hours in 12 hL and 24 h SD rats. Nonetheless, the final mean percentage of NREM sleep in these two groups of SD rats was always less than 4%. 3.2. NGFI-A expression in control rats Quantitative data referring to the distribution of NGFI-A mRNA are in Table 1.

M. Pompeiano et al.r Molecular Brain Research 46 (1997) 143–153

Control rats showed a distribution of the hybridization signals in accordance with the reported distribution of NGFI-A mRNA w44x. The strongest signals were seen in the cerebral cortex, particularly the piriform cortex, hippocampal formation Žgranule cell layer of the dentate

147

gyrus and pyramidal cell layer of all CA fields., olfactory bulb and anterior olfactory nucleus and the cerebellum. Intermediate-to-high levels of NGFI-A mRNA were seen in the caudate-putamen, amygdala Žbasolateral and central nuclei., claustrum, endopiriform nucleus and medial habe-

Fig. 1. NGFI-A mRNA expression increased in the cerebral cortex and decreased in the hippocampal formation after SD Žsee text for details.. Coronal X X X X Y sections correspond to plates 30–31 of w36x. A , B , C , D and D are representative sections of 3 hL, 6 hL, 12 hL, 12 hD and 24 h SD rats, respectively. A, B, C and D are the corresponding controls. CA1, field 1 of Ammon’s horn; CA2–3, field 2–3 of Ammon’s horn; DG, dentate gyrus; MHb, medial habenular nucleus; Par, parietal cortex; Pir, piriform cortex. Bar, 5 mm.

148

M. Pompeiano et al.r Molecular Brain Research 46 (1997) 143–153

X

X

Fig. 2. NGFI-A mRNA expression in the gigantocellular reticular nucleus showed a tendency towards a decrease after 3 hL SD ŽA and B . with respect to the corresponding controls ŽA and B., which appeared to be mainly due to the disappearance of strongly labeled cells present in the control sleeping rats. X X This is shown at two different levels, corresponding to plates 63 ŽA and A . and 68 ŽB and B . of w36x. 7, facial nucleus; Cb, cerebellum; Gi, gigantocellular reticular nucleus; py, pyramidal tract. Bar, 1 mm.

nular nucleus. Lower levels were seen in the septum, thalamus Žanterior nuclear group. and hypothalamus Žsuprachiasmatic, supraoptic, dorso- and ventromedial nuclei., colliculi, pontine nuclei and locus coeruleus. Signal intensity appeared to be higher in controls sacrificed during dark than during light hours, in accordance with previous data w39x. However, no attempt was made to make this comparison quantitative because of possible differences due to processing these tissues at different times. 3.3. NGFI-A expression in SD rats Nested ANOVA on the data from all four SD experiments Ž3 hL, 6 hL, 12 hL and 12 hDr24 h. gave similar results, with no significant variation found between replicates or between sides and with significant variation found between conditions within brain regions Ž3 hL: replicates,

F s 0.607, df s 1, P s 0.44, side, F s 0.155, df s 1, P s 0.69, condition nested within region, F s 142.868, df s 117, P s 0.0001; 6 hL: replicates, F s 3.236, df s 1, P s 0.07, side, F s 0.00004, df s 1, P s 1.00, condition nested within region, F s 331.075, df s 117, P s 0.0001; 12 hL: replicates, F s 0.708, df s 1, P s 0.40, side, F s 3.930, df s 1, P s 0.05, condition nested within region, F s 602.905, df s 117, P s 0.0001; 12 hDr24 h: replicates, F s 0.103, df s 1, P s 0.75, side, F s 0.004, df s 1, P s 0.95, condition nested within region, F s 339.682, df s 176, P s 0.0001.. The only exception to this pattern was a marginally significant variation between sides in the 12 hL data Ž P s 0.05.. This appeared to be due to greater variation of staining intensity than seen in the other experiments. Post-hoc tests failed to find any significant differences in staining between the right and left sides for any of the regions examined.

Fig. 3. NGFI-A mRNA expression increased in the cerebral cortex and caudate-putamen and decreased in the anterior nuclear group of the thalamus after 6 X hL SD ŽA . with respect to the corresponding controls ŽA.. Coronal sections correspond to plate 24 of w36x. AN, anterior nuclear group of the thalamus; Cg, cingulate cortex; CPu, caudate-putamen; Fr, frontal cortex; M, motor cortex; Par, parietal cortex; Pir, piriform cortex. Bar, 5 mm.

M. Pompeiano et al.r Molecular Brain Research 46 (1997) 143–153

149

X Fig. 4. NGFI-A mRNA expression decreased in the olfactory bulb after 12 hL SD ŽA . with respect to the corresponding controls ŽA.. OB, olfactory bulb. Bar, 2 mm.

SD resulted in significant changes in NGFI-A expression in many brain areas revealed by BonferronirDunn corrected post-hoc tests, as reported in Table 1. After 3 hL SD, rats showed increased NGFI-A mRNA levels in some cortical areas, such as the frontal, occipital, cingulate and perirhinal cortex, with respect to the controls ŽFig. 1A-AX .. A tendency towards an increase was seen also in other cortical areas and in the caudate-putamen ŽFig. 1A-AX .. A reduction of NGFI-A mRNA was seen in the granule cell layer of the dentate gyrus ŽFig. 1A-AX . and in the latero-dorsal nucleus of the thalamus. In the brainstem, a tendency towards a decrease of the hybridization signal was seen in the gigantocellular reticular nucleus ŽFig. 2., which appeared to be mainly due to the disappearance of strongly labeled cells present in the control sleeping rats. After 6 hL SD, NGFI-A expression increased with respect to controls in many cortical areas ŽFig. 1B-BX ., caudate-putamen ŽFig. 1B-BX , Fig. 3., claustrum and dorsal endopiriform nucleus. The hybridization signals were reduced in the granule cell layer of the dentate gyrus ŽFig. 1B-BX . and the anterior nuclear group of the thalamus ŽFig. 3.. A tendency toward a reduction was seen also in the pyramidal cell layer of the CA2–3 fields of the hippocampus ŽFig. 1B-BX . and in some other thalamic nuclei, particularly the medial and lateral habenular nucleus ŽFig. 3. and laterodorsal nucleus. After 12 hL SD, an increase of the hybridization signals was seen in some cortical areas, such as the frontal, parietal, temporal and perirhinal cortex ŽFig. 1C-CX ., and in the dorsal endopiriform nucleus. A tendency towards an increase was also seen in the caudate-putamen ŽFig. 1C-CX .. A decrease in NGFI-A mRNA levels was detected in the olfactory bulb ŽFig. 4., granule cell layer of the dentate gyrus and pyramidal cell layer of CA1 and CA2–3 fields ŽFig. 1C-CX ., dorsomedial hypothalamic nucleus and several nuclei of the thalamus Žmedial and lateral habenular nuclei, centromedial and centrolateral nuclei, rhomboid-reuniens nuclei, laterodorsal nucleus and ventroposterior nuclear group. ŽFig. 1C-CX .. After 12 hD SD, NGFI-A mRNA increased in the

frontal and temporal cortex and in the anterior olfactory nucleus. A tendency toward an increase was also seen in other cortical areas Žparietal, occipital and cingulate cortex; Fig. 1D-DX . while a tendency toward a decrease was still detectable in the granule cell layer of the dentate gyrus ŽFig. 1D-DX .. After 24 h SD, the hybridization signal increased, with respect to controls, in the anterior olfactory nucleus and decreased in the subbrachial nucleus in the brainstem. The signal showed a tendency toward a decrease in the granule cell layer of the dentate gyrus ŽFig. 1D-DY ., laterodorsal nucleus of the thalamus and superior colliculus.

4. Discussion By using in situ hybridization histochemistry, we demonstrated that periods of SD up to 24 h result in marked changes in NGFI-A expression in several brain areas. Interestingly, both increases and decreases of NGFIA mRNA levels were observed, depending upon the area. 4.1. Effects of different SD periods While 3 hL SD were sufficient to induce changes in NGFI-A expression in specific brain areas, the strongest and most diffuse increase, particularly in the cerebral cortex and caudate-putamen, was seen after 6 hL SD. After 12 hL SD, such an increase was not so evident, while a clear reduction in NGFI-A expression was seen in areas, such as the olfactory bulb, CA1–3 fields of the hippocampus and many nuclei of the hypothalamus and thalamus. In the anterior nuclear group of the thalamus, however, NGFI-A expression decreased only at shorter SD periods Ž6 hL.. NGFI-A expression was generally much less affected by SD performed during the dark hours. If we compare 12 hL and 12 hD SD, the effects are restricted to a few areas in the latter case. This may in part be due to the fact that NGFI-A expression during the night, when rats are active, is higher than during the day w39x and this could result in a

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plateau effect at least in some areas. However, in some cerebrocortical areas the increase of NGFI-A mRNA seen after 12 hD SD was also very strong and comparable to that seen after 6 hL SD. In the anterior olfactory nucleus, NGFI-A mRNA increased only after 12 hD and 24 h SD. The 24-h SD period was the least effective on NGFI-A expression. All SD periods performed during the day were associated with a reduction of NGFI-A mRNA levels in the granule cell layer of the dentate gyrus. The SD performed during the night, however, showed a clear tendency toward a decrease of NGFI-A expression in this latter region. The observation of changes in NGFI-A mRNA expression after 3 hL SD is in agreement with the temporal dynamics of NGFI-A mRNA induction reported in other studies both in vivo and in vitro Že.g. w29x.. The decreased induction observed after 12 hL SD with respect to shorter SD periods is also in agreement with in vitro studies w29x. A complex transcriptional regulation of the expression of some IEGs by their own protein products has been documented. For example, c-fos and c-jun expression are negatively and positively regulated, respectively, by the corresponding protein products w17x. Two NGFI-A consensus sequences are found in the NGFI-A promoter region, through which NGFI-A could down-regulate its own expression w7x. This would result in a refractory period, which would also explain why 24 h SD were less effective than 12 hD SD.

4.2. Comparison with other studies Other authors have examined the effects of SD on NGFI-A expression in the rat brain. Our results are consistent with those of O’Hara et al. w32x, who demonstrated with Northern blots that NGFI-A increased in the cerebral cortex of SD rats with respect to controls and that the increase was stronger after 6 h than after 3 h SD. Landis et al. w23x examined NGFI-A expression in rats subjected to 10 days of total SD. They did not found any significant difference in NGFI-A mRNA levels in whole brain as detected with Northern blotting. This would be in accordance with our observation that longer Ž24 h. SD periods were less effective than shorter SD periods in affecting NGFI-A expression. By using immunocytochemistry, Landis et al. found an increase in NGFI-A protein levels in lateral habenula, superior colliculus, ventral periaqueductal gray and dorsal raphe and a decrease in the outer lamina of the posterior cingulate cortex and dentate gyrus in SD rats with respect to controls. Our data agree with those of Landis et al. for what concerns the dentate gyrus, while for the other regions, we observed no change or a change in the opposite direction. This discrepancy could be due to a specific effect of the much longer SD periods used by these authors, which we did not examine.

4.3. SD and stress Stress represented a major concern in our studies of the effects of SD on gene expression. We used a SD procedure Žgentle handling. which has been demonstrated not to affect plasma levels of corticosterone or other peripheral indicators of stress w31,52x. In a previous study w9x, we observed that c-fos expression increased after SD in the same areas as after spontaneous wakefulness, while it did not change in other areas in which it has been described to increase after stress w5,6x. From these observations, we concluded that stress did not play an important role in our experiments. As further support for this conclusion, NGFIA expression decreased in CA1 after SD, while in the same area it increased after stress w45x.

4.4. Comparison between NGFI-A and c-fos It has been suggested that changes in NGFI-A expression may reflect changes in physiological activation more rapidly than changes in c-fos w56x. This seems to hold true during spontaneous wakefulness. In fact, after only 30 min of spontaneous waking, increases in both NGFI-A mRNA and protein were seen in some cerebrocortical regions, while c-fos expression increased only at the mRNA level w39x. In the SD protocol, NGFI-A mRNA seems to vary less promptly than c-fos. In fact, the most effective SD period for c-fos induction was 3 hL SD w9x, while for NGFI-A it was 6 hL SD. On the other hand, if we compare the effects of different SD periods on c-fos w9x and NGFI-A induction, our results are in good agreement with the observation that the NGFI-A mRNA decay rate is slower than that of c-fos mRNA w29x. In the cerebral cortex and caudate-putamen, c-fos expression increased mainly after 3 hL SD, while NGFI-A expression was increased at all SD periods performed during the light hours. In the hippocampus, an increase in c-fos expression was observed at the shortest SD periods, while NGFI-A mRNA decreased in the same regions, especially in the dentate gyrus, at all SD periods. c-fos expression increased in both olfactory bulb and anterior olfactory nucleus at the shortest SD periods, 3 hL and 6 hL, respectively. NGFI-A mRNA decreased in the olfactory bulb and increased in the anterior olfactory bulb, but only at longer SD periods. In the medial preoptic area of the hypothalamus, c-fos expression increased, while NGFI-A mRNA levels did not change. For some hypothalamic and thalamic nuclei, c-fos mRNA increased after 3 hL SD and decreased after 6 hL SD, probably due to shut-down mechanisms, while NGFI-A mRNA decreased after 12 hL SD. c-fos expression increased following SD in several nuclei of the brainstem while NGFI-A mRNA levels in case decreased in few areas.

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The different temporal and spatial patterns of NGFI-A and c-fos expression observed in our SD studies can be explained by considering that these two IEGs do not necessarily respond in the same ways to all stimuli. In addition, c-fos and NGFI-A expression may be related to different stimuli present in the SD condition. 4.5. Comparison between spontaneous and forced wakefulness NGFI-A expression in 6 hL SD was comparable to that observed in waking-dark ŽW-D. rats, which were killed at 02:00 h and had been spontaneously awake for 6 h before sacrifice w39x. In both conditions, NGFI-A mRNA increased in the cerebral cortex, caudate-putamen, claustrum and dorsal endopiriform nucleus and it decreased in the anterior nuclear group of the thalamus with respect to the corresponding sleeping controls. 6 hL SD rats exhibited greater changes in NGFI-A expression, but basically in the same areas, than waking-light ŽW-L. rats, which were also sacrificed at 14:00 h but had been awake for only 30 min before sacrifice w39x. These observations suggest that in these areas NGFI-A may mark neuronal populations which function in a common mode during either forced or spontaneous wakefulness, irrespective of circadian factors. In a previous study, we reported that when our tactile stimulation protocol for SD was applied unilaterally, the pattern of Fos labeling in 3 hL SD rats was similar to that observed when the stimulation was applied bilaterally Žas normally done. and that no asymmetries in c-fos expression were noticed in the somatosensory cortex w9x. This would suggest that changes in c-fos expression in this area are due to wakefulness per se rather than being a mere effect of the SD procedure itself. The same was observed for NGFI-A expression Žunpublished results.. The fact, however, that spontaneous and forced wakefulness are qualitatively different is mirrored by the observation of clear differences in the NGFI-A expression pattern between these two conditions. Interestingly, this was less evident for c-fos expression, thus, suggesting a different role for these two genes. In the hippocampal formation, NGFI-A expression decreased in the dentate gyrus and in the CA fields after SD. On the other hand, NGFI-A expression increased, at least in terms of protein levels, during spontaneous wakefulness in these areas. Since NGFI-A expression may be involved in memory trace formation w1,42,44x, the decrease of NGFI-A mRNA levels in the dentate gyrus may, thus, represent a molecular correlate of memory impairment seen following SD w21,47x. In the olfactory bulb, NGFI-A mRNA increased in both W-L and W-D animals w39x, while it did not show any change after SD, except for a decrease after 12 hL SD. Even if IEG expression in this area may be related to sniffing activity per se w15x rather than to some form of olfactory memory w4x, the fact that rats show spontaneous

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sniffing behaviour during the night when they are mainly awake and that SD may impair this activity may at least in part account for our data. In the anterior olfactory nucleus, which is strongly and reciprocally connected to the olfactory bulb w49x, NGFI-A mRNA levels increased in W-D rats and the same was seen for SD periods performed during the night. NGFI-A expression increased after spontaneous wakefulness, but not after SD, in the medial preoptic area, a region which has been involved in the regulation of sleep Že.g. w20,28x.. We observed that the expression of c-fos increased after both spontaneous wakefulness w39x and SD w9x in this area. Local injections of c-fos antisense oligonucleotides led to a marked reduction of sleep the day after the injections w8x, suggesting that c-fos expression in the medial preoptic area is part of the chain of events involved in the homeostatic regulation of sleep. NGFI-A in the medial preoptic area, thus, appears to be involved in different processes than c-fos. 6 h of either spontaneous or forced wakefulness resulted in a decrease in NGFI-A expression in the anterior nuclear group of the thalamus with respect to the corresponding sleeping control sacrificed at 14:00 h. Alternatively, it may be said that 6 h of sleep induce NGFI-A expression. Interestingly, the anterior thalamic nuclei are among the few thalamic nuclei that do not receive afferents from the reticular thalamic nucleus, at least in the cat w48x and that do not display spindle rhythms. These nuclei strongly project to the whole limbic cortex w46x and are interposed in a circuit that transmits u waves to the cingulate cortex w48x. In the thalamus, NGFI-A mRNA decreased also in the laterodorsal nucleus especially at 12 hL SD. In this nucleus, NGFI-A expression increased during spontaneous wakefulness w39x. Interestingly, the laterodorsal nucleus also projects to the limbic cortex w55x. 4.6. Possible functional significance of NGFI-A expression after SD Regulation of gene expression is a complex and highly integrated process Že.g. w11,16x.. Extracellular stimuli generate signals leading to long-term changes in cell behaviour by changing programmes of gene expression. Several stimuli can induce IEG expression w17x and in many cases the activation of a single intracellular signal pathway has been demonstrated to be sufficient. Constitutive expression of NGFI-A in neocortex, but not in hippocampus, appears to be dependent upon tonic activity of the noradrenergic input from the locus coeruleus, while in the striatum it would mainly require activity of the dopaminergic system w17x. We recently hypothesized that the activity of neuromodulatory systems with diffuse projections, such as the monoaminergic systems, may coordinate not only changes in cell excitability and activity but they may also influence programmes of gene expression occurring during the

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sleep-waking cycle in many brain regions w53x. This hypothesis was mainly based on the following evidence: Ž1. dramatic changes in the expression of IEGs, and possibly of other genes, are seen between waking and sleep Žsee references in w53x.; Ž2. the activity of such modulatory systems also changes dramatically during the sleep-waking cycle w48x; Ž3. the activity of these systems influences the expression of IEGs, and possibly of other genes, through specific receptor subtypes Žsee references in w53x.. Supporting this hypothesis, we recently demonstrated that both c-fos and NGFI-A expression in the cerebral cortex during spontaneous wakefulness and SD is indeed dependent on the intactness of the noradrenergic projection from the locus coeruleus w10,54x. The increased NGFI-A expression observed in cerebral cortex after either spontaneous w39x and forced wakefulness is consistent with the higher discharge rate of locus coeruleus neurons during wakefulness w48x. On the other hand, the decreased efficacy of longer SD periods, particularly 24 h, in sustaining an increased NGFI-A expression may be related to the observation of a decrease in the waking discharge of REM-off, presumably noradrenergic locus coeruleus neurons, after comparably long periods of REM SD w27x. The lower efficacy of longer SD periods in inducing NGFI-A expression may, thus, represent a molecular correlate of the finding of Mallick et al. w27x which they relate to the increased sleep pressure and the decreased vigilancerarousal seen during SD. The demonstration of changes in IEG expression correlated with different sleep and waking states naturally leads to the problem of the identification of the pattern of late genes, whose expression would also be orchestrated and which could be directly involved in sleep regulation and function. In a database search, 164 different potential target genes for NGFI-A have been found w24x. Techniques, such as subtractive hybridization w41x and mRNA differential display w26,40x, will probably be of great help in systematically tackling this problem.

Acknowledgements We thank Evan Balaban for critically reading the manuscript and for help with the statistical analysis and two anonymous reviewers for helpful comments. We are grateful to Ottavio Pompeiano, who supported this work. Giovanna Bresciani provided excellent animal care and Piero Bertelli and Mario Pardini excellent technical assistance. This work was supported by the 1992 European Sleep Research Society-Synthelabo Grant, by grants of the Ministero dellX Universita’ e della Ricerca Scientifica e Tecnologica Ž40% and 60%., the Agenzia Spaziale Italiana ŽASI 95-RS-53. and the National Institute of Neurological and Communicative Disorders and Stroke Research ŽNS 07685-26..

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