Theme : Dreams

Alterations in Monoamine Neurotransmitters and Dendritic Spine Densities at the Medial Preoptic Area after Sleep Deprivation

Vijay Ramesh 1, Madepalli K. Lakshmana 2, B.S. Shankaranarayana Rao 2, Trichur R. Raju 2 and Velayudhan Mohan Kumar1
1Department of Physiology, All India Institute of Medical Sciences, New Delhi 110 029, India and 2Department of Neurophysiology, National Institute of Mental Health and Neurosciences, Bangalore 560 029, India
Abstract
The experiments were conducted on 24 adult male Wistar rats to find out the alterations in the levels of monoamines and dendritic spine densities in the medial preoptic area and cortex after total sleep deprivation. Noradrenaline was reduced in the medial preoptic area, though there was no significant change in the cortex. Dopamine and serotonin were decreased both in the medial preoptic area and in the cortex. Dendritic spine counts in the medial preoptic area and the motor cortex were increased after total sleep deprivation. Enhanced release of the monoamines and their subsequent breakdown during sleep deprivation could be responsible for the decreased levels of the transmitters. An increase in synaptic activity, resulting in the enhanced release of the transmitters, might be responsible for the increased spine density after total sleep deprivation. Localized changes in noradrenaline levels at the medial preoptic area suggest its involvement in sleep genesis and maintenance, though its possible contribution to other functions like thermoregulation and reproduction cannot be ruled out. As the available literature does not indicate a role for serotonin and dopamine at the medial preoptic area in sleep regulation, these changes may represent their participation in non-sleep functions.

Current Claim: Increased sleep pressure, resulting from total sleep deprivation, causes enhanced release of noradrenaline and other monoamines and an increase in the spine density at the medial preoptic area.

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Suppression of sleep and reduction of sleep episode duration, produced by the lesion of the medial preoptic area (mPOA), showed its importance in the regulation of sleep, especially the maintenance of sleep (Nauta, 1946; Sterman and Clemente, 1962; John et al., 1994; John and Kumar, 1998). Sleep and EEG synchronization, induced by electrical and thermal stimulation of this area, indicate its role in the genesis of sleep (Parmeggiani et al., 1974; Benedek et al., 1979). Alterations in single cell activities recorded from this area suggest its involvement not only in regulation of slow wave sleep but also REM sleep (Findlay and Hayward, 1969; Koyama and Hayaishi, 1994; Alam et al., 1995). It was hypothesized, on the basis of responses elicited by local administration of transmitters and blockers, that the adrenergic afferents in the mPOA control sleep (Kumar et al., 1984, 1986, 1993; Datta et al., 1988; Ramesh et al., 1995; Ramesh and Kumar, 1998).
The mPOA receives noradrenergic, dopaminergic and serotonergic afferent inputs (Anden et al., 1966; Palkovits and Zaborszky, 1979). Though the noradrenergic fibres have been shown to play a hypnogenic role at the level of the mPOA, the role played by the other monoaminergic fibres in the regulation of sleep-wakefulness (S-W) is still debated (Kumar et al., 1993; Ramesh et al., 1995; Sood et al., 1997; Ramesh and Kumar, 1998). The noradrenergic system is shown to be essential for sleep as lesioning the locus ceruleus with DSP-4 produced a significant decrease in the sleep rebound after sleep deprivation (Gonzalez et al., 1996). It was suggested that the noradrenergic inputs to the mPOA promote sleep as destruction of the noradrenergic fibres in the mPOA produce an increase in wakefulness (Kumar et al., 1993). Sleep induced by the local administration of adrenergic agents further supports this contention (Kumar et al., 1993; Ramesh et al., 1995; Ramesh and Kumar, 1998). Destruction of dopaminergic fibres at the mPOA did not produce any change in S-W (Kumar et al., 1993). Though serotonin (5-HT) has been implicated in the genesis of sleep, local application of this transmitter at the mPOA did not produce any alteration in S-W (Yamaguchi et al., 1963; Marley and Whelan, 1975; Datta et al., 1987). The probable involvement of these monoaminergic systems at the level of the mPOA in the regulation of sleep could be further investigated by assessing the levels of noradrenaline (NA), dopamine (DA) and 5-HT in this area after sleep deprivation. It has been reported that the deprivation of sleep produced alterations in NA, DA and 5-HT levels in various regions of the brain including the hypothalamus (Asikainen et al., 1995; Porkka-Heiskanen et al., 1995; Farooqui et al., 1996). If some of these monoamines are involved in the regulation of sleep at the level of the mPOA, their levels may be altered after sleep deprivation. At the same time, the involvement of monoamines cannot be ruled out if their levels are not changed. Other functions like thermoregulation, food intake and sex drive, which may be altered by sleep deprivation (Bhanot et al., 1989; Rechtschaffen et al., 1989; Verma et al., 1989a), can also contribute to the transmitter change.

Morphological study of dendritic spines could also give information about the altered transmitter activity during sleep deprivation. Excessive synaptic activity has been shown to increase the spine density in the neocortex and mPOA (Globus and Scheibel, 1966; Valverde, 1967; Sanchez-Toscano et al., 1991). If there is any alteration in the release of one or more of these monoaminergic transmitters during sleep deprivation, it is likely to produce an alteration in the spine density at the mPOA, though the contribution of some other transmitters, whose levels were not estimated, cannot be ruled out. So, in this study, in addition to the levels of monoamines, the spine density of the neurons in the mPOA was studied after total sleep deprivation (TSD). The results from the sleep deprived animals were compared with those of the control groups which were not subjected to TSD. The transmitter levels and the morphology of the motor cortex were also studied to get an idea about the regional specificity of the induced changes.

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Experiments were conducted on 24 male adult Wistar rats weighing between 250 and 275 g. They were housed separately in an animal room with the ambient temperature maintained at 26±1°C and light on period from 05:00 h to 19:00 h. Food and water were provided ad libitum. The rats were randomly divided into four groups of six each. Two groups were used as controls and the other two as experimental groups. All animals in the experimental groups were allowed to get acquainted with the sleep deprivation chamber. They were then subjected to 48 h TSD, starting from 19:00 h, by placing them in a drum (cage) of 25 cm diameter rotating at the speed of 1 revolution/45 s (Borbely and Neuhaus, 1979). The rats tumbled for a few minutes in the beginning of TSD as the drum rotated. Before an hour, all the rats learned to avoid tumbling by moving to the other side of the cage in the direction opposite to that of rotation. They rested there until the rotating cage brought them again to an angle at which they would tumble. At that critical angle of tumbling they again moved to the other side. There was not much difference in the time taken by the rats to learn to avoid tumbling. The rats had free access to food and water inside the TSD chamber.
The experimental group of rats were decapitated immediately after TSD and the mPOA were bilaterally dissected out by gently teasing out the optic chiasma. A rostral coronal cut at the level of the bifurcation of the anterior cerebral artery, a parallel cut approximately 1 mm caudal to this, two sagittal cuts 0.5 mm lateral to the midline, and a final 1 mm deep undercut were given to remove the mPOA. The motor cortices corresponding to the area 4 and to the lateral field of the agranular cortex (Donoghue and Wise, 1982; Neafsey et al., 1986; Vogt and Miller, 1988), were dissected out. Rats from the control group were also sacrificed at identical timings, and the mPOA and motor cortex were dissected out as mentioned above. The mPOA and motor cortex from one control and one experimental group were weighed and prepared for the estimation of monoamines and their metabolites. The brain tissues from the other two groups were processed for the assessment of dendritic morphology in 100-120 µm thick Golgi-stained sections (Hammer et al., 1981; Gundappa and Desiraju, 1988).

Estimation of monoamines
The mPOA and motor cortex were homogenized and the monoamines and some of their metabolites were estimated using HPLC (ERC-8710, Erma Optical Works, Japan) coupled to a fluorescence spectrophotometer (Hitachi, model 650-40, Japan) at the excitation wavelength of 280 nm and emission wavelength of 315 nm, keeping the slit width at 10 for both excitation and emission (Lakshmana and Raju, 1997). NA, DA and 5-HT in the tissue were quantified by comparing peak heights in elution profiles of samples with known standards. 5-hydroxy-tryptophan (5-HTP) coeluted with the metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), and tryptophan (TRP) coeluted with 5-hydroxyindole acetic acid (5-HIAA). The peak heights of coeluted samples were compared with their respective standards (Lakshmana and Raju, 1997). The levels of monoamines and other substances of the experimental group were compared with the control group by two-tailed student's t-test (Lakshmana and Raju, 1994). The ratio of 5-HIAA/5-HT and DOPAC/DA were also compared between control and experimental groups, using the same test, by taking the coeluted values mentioned above.

Dendritic spine density
The number of dendritic spines of six neurons from the mPOA and the motor cortex, taken from different sections from each rat, were counted. The spine densities of 72 mPOA neurons with dendrites of a length of more than 50 µm from the soma (Fig. 1), and 72 large pyramidal cells of the fifth layer of the motor cortex were counted. All neuronal types were confirmed by counter staining with 2% cresyl violet. The neurons with overlapping processes from neighboring impregnated cells were not selected for the count. The standard criteria were used for selection of the neurons (Shankaranarayana Rao et al., 1993).

The spines were observed and counted along the dendritic margins at a magnification of 1500 in oil immersion as described elsewhere (Gundappa and Desiraju, 1988). The spines were counted on successive dendritic segments of 10 µm from the soma up to 50 µm lengths for mPOA neurons, and on successive dendritic segments of 15.2 µm up to 76 µm, for motor cortex neurons. The spines of the pyramidal cells were counted from the main shafts of the basal dendrites, their primary and secondary branches. The spines of the main shafts of the apical dendrites, their oblique shafts and primary branches were also counted (Fig. 2). Since the mPOA neurons were small and heterogenic the spines were counted from all dendrites without any subdivision. The slides were coded and randomly assigned. The spines were counted by a double-blind method to overcome the observer bias. Brain tissues were sectioned in the coronal plane and were sampled from both sides. Counts from the experimental groups were compared with the control group using two-tailed student's t-test (Sunanda et al., 1995).

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Figure 1

Figure 2

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Figure 6

The basal levels of NA, DA and 5-HT were much higher at the mPOA as compared to the motor cortex in both TSD and control groups. NA was reduced in the mPOA after TSD, though there was no significant change in the cortex (Fig. 3). DA and 5-HT levels were lowered in the mPOA and the motor cortex. All three biogenic amines were more significantly reduced in the mPOA than in the cortex after TSD. DOPAC and 5-HTP showed an increase in the mPOA, though the change was not significant for 5-HIAA and TRP (Fig. 3). Changes in the levels of these substances at the cortex were not statistically significant. The ratio of 5-HIAA/5-HT in the mPOA after TSD (1.80±0.12) was significantly higher (p<0.001) than in the control group (0.99±0.08). Similarly, the ratio of DOPAC/DA after TSD (2.61±0.19) in the mPOA was also higher (p<0.001) than in the control group (1.74±0.11). In the cortex, the ratio of 5-HIAA/5-HT in the experimental group (0.88±0.17) was not significantly different from that in the control group (0.60±0.13). The ratio of DOPAC/DA in the cortex in the TSD group (1.42±0.15) was higher (p<0.05) than in the control group (1.07±0.29).
Studies on two other groups of rats showed that the spine densities were higher at distal parts of the dendrites of both the mPOA and cortical neurons. The total number of spines in the mPOA neurons were found to be significantly higher in the TSD group as compared to the control group (Fig. 4). The spine counts from the TSD animals were significantly higher at some branches of apical and basal dendrites, especially at distal segments, of the cortical neurons (Figs. 5 and 6).

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TSD for 48h had led to a decrease in the monoamines, which was more drastic in the mPOA than in the motor cortex. There was an increase in the number of dendritic spines at the mPOA and the motor cortex.
Monoamines
NA concentration was decreased in the mPOA, though no significant alteration was found in the cortex. According to an earlier report, NA concentration was lower in the posterior hypothalamus, neocortex and hippocampus after REM sleep deprivation for 24h (Porkka-Heiskanen et al., 1995). However, according to another report, the NA concentrations in frontal and parietal cortices remained unchanged even after REM sleep deprivation for 96 h (Farooqui et al., 1996). On the other hand, TSD for 4h produced no change in NA concentrations in the hypothalamus, frontal cortex, hippocampus, brain stem and olfactory bulb (Asikainen et al., 1995). Thus, the TSD for 48h produced a significant decrease in the NA, which is by and large localized to the mPOA.

It has been suggested that the enhanced release of NA, resulting from an increased impulse activity of the adrenergic system, led to a depletion of this transmitter in the nerve terminals (Porkka-Heiskanen et al., 1995). So, it could be taken to indicate that the increased sleep pressure during TSD caused an enhanced release of NA at the mPOA. Highly significant decreases of NA at the mPOA, without any significant change in the cortex could be taken as further support to the contention that the noradrenergic fibres play a hypnogenic role at the level of the mPOA (Kumar et al., 1993; Ramesh et al., 1995; Ramesh and Kumar, 1998). Though the noradrenergic fibers projecting to the mPOA have been shown to be involved in the induction of sleep, these fibres have also been implicated in the arousal mechanism at the cortical level (Jones et al., 1973; Kumar et al., 1993). NA administration at the mPOA has also been shown to alter sex behavior and thermoregulation (Datta et al., 1988; Mallick et al., 1996). Sleep deprivation has also been shown to affect these functions (Rechtschaffen et al., 1989; Verma et al., 1989a). So, the possible involvement of these functions in the observed alteration in NA cannot be ruled out. It has also been suggested that stress may cause increased release of NA from the terminals in the neocortex and posterior hypothalamus, eventually leading to a decrease in NA concentration in the terminals (Porkka-Heiskanen et al., 1995).

The decrease in 5-HT in the mPOA after TSD could be taken to indicate an increased turnover of 5-HT. An increase in the turnover of 5-HT in the hypothalamus has been suggested, as the 5-HIAA/5-HT ratio was significantly increased after TSD (Asikainen et al., 1997). In this study, this ratio was also found to be significantly higher in the mPOA after TSD, though there was no change in the motor cortex. However, much emphasis cannot be placed on this finding, as the 5-HIAA values were assumed on the basis of peaks which coeluted with TRP. The decrease in 5-HT levels in the cortex is comparable to that shown in some of the earlier reports. The 5-HIAA/5-HT ratio was significantly increased after TSD not only in the hypothalamus and the frontal cortex, but also in the hippocampus and brain stem, indicating an increased 5-HT turnover in all these regions (Asikainen et al., 1997). According to their earlier report, there were no changes in 5-HT turnover in the frontal cortex and olfactory bulb in hamsters after TSD for 4h (Asikainen et al., 1995). After REM sleep deprivation the concentration of both 5-HT and HIAA were shown to be reduced in the frontal and parietal cortices (Farooqui et al., 1996). So, the 5-HT turnover is probably increased throughout the brain after TSD, though it is significantly increased in the mPOA.

The DA levels were reduced both at the mPOA and the cortex after TSD, though the changes in the level of the metabolite DOPAC were significant only in the mPOA. An increased level of DOPAC along with a decrease in DA could be taken to indicate an increased turnover of DA at the mPOA. The DOPAC/DA ratio was also increased at the mPOA and cortex. A great deal of importance cannot be given to this finding as it is based on the height of the peak of DOPAC which coeluted with 5-HTP. According to another report, DA and DOPAC concentrations were elevated in the hypothalamus after TSD for 4h (Asikainen et al., 1995). The concentration of DA, DOPAC and homovanillic acid remained unchanged in the frontal and parietal cortices after REM sleep deprivation for 96h (Farooqui et al., 1996).

It is difficult to explain the functional importance of a decrease in DA and 5-HT levels in the mPOA and cortex after TSD. Activation of hypothalamic-pituitary-adrenal axis and stimulation of various neurotransmitter systems in response to stress have been suggested as a factor in the increase in dopamine metabolites in the hypothalamus (Iuvone et al., 1977; Rees and Gray, 1984; Gilad et al., 1987; Farooqui et al., 1996). These transmitters have also been implicated in many other functions, such as thermoregulation and reproduction at the level of the mPOA (Rodriguez et al., 1984; Datta et al., 1987; Verma et al., 1989a; Mallick et al., 1996). These functions are also altered after sleep deprivation (Verma et al., 1989b; Franken et al., 1993). The altered levels of DA and 5-HT might be playing a role in the alteration of these functions.

A decrease in the levels of monoamines of TSD rats could indicate either a lower level of release of these transmitters or their depletion due to synaptic over-activity. A decrease in the monoamine levels, along with an increase in their products of metabolism, may indicate the second possibility mentioned above. A study in which all the metabolites of the monoamines are estimated, or one in which actual release of these neurotransmitters are measured by microdialysis or voltametric techniques, would provide a better answer to this question.

Dendritic spine density
An increase in synaptic activity has also been shown to elevate the spine number in the central nervous system (Globus and Scheibel, 1966; Valverde, 1967). Earlier studies have shown an increase in spine densities in different brain regions after exposing the animals to different environmental conditions (Gundappa and Desiraju, 1988; Mahajan and Desiraju, 1988; Shankaranarayana Rao et al., 1993; Sunanda et al., 1995). Long-term electrical stimulation of the presynaptic pathways to the pyramidal cells in adult cats produced an increase in spine density (Rutledge et al., 1974). An increase in spine density in the mPOA neurons of the rats subjected to prolonged isolation stress has been attributed to enhanced synaptic activity (Sanchez-Toscano et al., 1991). Though the altered levels of monoamines could be playing an important role in the morphological changes, the role of other transmitters cannot be ruled out. REM sleep deprivation significantly increased the levels of glutamate and glutamine (Bettendorff et al., 1996). The altered levels of these excitatory amino acids, and possibly many other transmitters, may also contribute to the post synaptic changes.

The possible contribution of some induced stress to the observed increase in spine densities cannot be ruled out (Sunanda et al., 1995). However, sleep deprivation in the rotation cylinder (1 rotation/45 s) produced a minimum amount of stress and physical exercise for the animal (Borbely and Neuhaus, 1979).

Thus, enhanced afferent input signals due to sustained sleep pressure in the TSD rats might have produced an increased release (and subsequent breakdown) of the monoamine transmitters in the brain, particularly in the mPOA. A significant decrease in NA, which was found only in the mPOA, provides further support for the claim that it plays an important role in sleep regulation in this region. The increased synaptic activity produced by an enhanced release of monoamines, especially NA, could be responsible for the increased spine density in the mPOA.

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This study was supported by the Indian Council of Medical Research.

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