**3. Results**

#### **3.1. Silver impregnation technique**

Topographically, the human hypothalamus is located between the lamina terminalis anteriorly and the posterior commissure and the posterior edge of the mammillary bodies, posteriorly. By rapid Golgi staining, the Golgi-Nissl method, and the other silver impregnation techniques, we could visualize the hypothalamic nuclei entirely and clearly. However, we focused our detailed description and measurement mostly on the suprachiasmatic (SCN), the supraoptic (SON), and the paraventricular nuclei (PVN).

The morphological and morphometric study of the hypothalamic nuclei revealed a substantial decrease of the number of neurons and an impressive loss of dendritic branches in the brains of the patients who suffered from AD (**Figures 1** and **2**), as compared with normal controls (**Figures 3** and **4**). Abbreviation of the dendritic arborization was prominent mostly in the neurons of suprachiasmatic nucleus (SCN). The dendritic alterations were associated with marked decrease in the number of dendritic spines (**Figures 5** and **6**) in comparison with the normal control brains (**Figure 7**). The same morphological alterations concerning the dendritic branches and the spines were also observed in the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus in AD (**Figure 8**).

The morphometric estimation of the dendritic spines of neurons of the SCN and SON revealed a dramatic decrease of spines in AD brains, in comparison with normal controls (**Table 2**).

paraventricular nuclei (PVN) of the hypothalamus in AD brains, in correlation with normal controls. Considerable decrease in spine density was mainly noticed in the secondary and tertiary dendritic branches, which was particularly prominent in the suprachiasmatic nucleus. Small spines and giant spines were also observed in a large number of neurons of the supra-

**Figure 2.** Neuron of SCN of the hypothalamus in a case of AD. The loss of the dendritic branches is obvious. Golgi

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In a considerable number of dendritic profiles, in the suprachiasmatic and the paraventricular nuclei, the mitochondria demonstrated marked morphological alterations, consisted of wide size diversity, disruption of the cristae, and accumulation of fibrillary material (**Figure 8**).

chiasmatic nucleus. Many giant spines included large multivesicular bodies.

**Figure 1.** Neuron of the SCN in AD brain. Golgi staining, 1200×.

staining Mag. 1200×.

#### **3.2. Electron microscopy**

Detailed study on electron microscope demonstrated substantial morphological changes of the dendritic arbors, concerning mostly the secondary and tertiary dendritic branches, in a substantial number of neurons of the suprachiasmatic (SCN), supraoptic (SON), and

**Figure 1.** Neuron of the SCN in AD brain. Golgi staining, 1200×.

method of concentric circles [64], which were drawn, at intervals of 15 μm, centered on the soma of the neuron. The dendritic spines were counted on three segments of the dendritic field. Thus, we calculated those, which were located: (a) on primary dendrite, 20–30 μm in length; (b) on the secondary dendrite, 20–30 μm in length; and (c) on the tertiary dendrite, 40–50 μm in length. In electron microscopy, we performed stereological analysis following the Nyengaard [65] and West [66, 67] principles. The number, the length, the total surface area, the volume, the circulatory ratio, and the spatial distribution of mitochondria [68] were precisely counted and

We also estimated the mean nuclear area, the dendritic profiles [70], the total number of the dendritic spines per dendritic segment, the pre- and post-synaptic components [71–73], and

The statistical analysis of the data was evaluated by Student t tests. p-Values below 0.05 were considered statistically significant, and those below 0.01 were considered as highly significant.

Topographically, the human hypothalamus is located between the lamina terminalis anteriorly and the posterior commissure and the posterior edge of the mammillary bodies, posteriorly. By rapid Golgi staining, the Golgi-Nissl method, and the other silver impregnation techniques, we could visualize the hypothalamic nuclei entirely and clearly. However, we focused our detailed description and measurement mostly on the suprachiasmatic (SCN), the

The morphological and morphometric study of the hypothalamic nuclei revealed a substantial decrease of the number of neurons and an impressive loss of dendritic branches in the brains of the patients who suffered from AD (**Figures 1** and **2**), as compared with normal controls (**Figures 3** and **4**). Abbreviation of the dendritic arborization was prominent mostly in the neurons of suprachiasmatic nucleus (SCN). The dendritic alterations were associated with marked decrease in the number of dendritic spines (**Figures 5** and **6**) in comparison with the normal control brains (**Figure 7**). The same morphological alterations concerning the dendritic branches and the spines were also observed in the supraoptic (SON) and paraven-

The morphometric estimation of the dendritic spines of neurons of the SCN and SON revealed a dramatic decrease of spines in AD brains, in comparison with normal controls (**Table 2**).

Detailed study on electron microscope demonstrated substantial morphological changes of the dendritic arbors, concerning mostly the secondary and tertiary dendritic branches, in a substantial number of neurons of the suprachiasmatic (SCN), supraoptic (SON), and

estimated as well as the cisternae and vesicles of the Golgi apparatus [69].

the number of synaptic vesicles per presynaptic terminal [73].

supraoptic (SON), and the paraventricular nuclei (PVN).

tricular nuclei (PVN) of the hypothalamus in AD (**Figure 8**).

**3. Results**

80 Hypothalamus in Health and Diseases

**3.1. Silver impregnation technique**

**3.2. Electron microscopy**

**Figure 2.** Neuron of SCN of the hypothalamus in a case of AD. The loss of the dendritic branches is obvious. Golgi staining Mag. 1200×.

paraventricular nuclei (PVN) of the hypothalamus in AD brains, in correlation with normal controls. Considerable decrease in spine density was mainly noticed in the secondary and tertiary dendritic branches, which was particularly prominent in the suprachiasmatic nucleus. Small spines and giant spines were also observed in a large number of neurons of the suprachiasmatic nucleus. Many giant spines included large multivesicular bodies.

In a considerable number of dendritic profiles, in the suprachiasmatic and the paraventricular nuclei, the mitochondria demonstrated marked morphological alterations, consisted of wide size diversity, disruption of the cristae, and accumulation of fibrillary material (**Figure 8**).

**Figure 3.** Neuron of the SCN of the hypothalamus of a normal brain aged 75 years.

**Figure 4.** Neuron of the SON of the hypothalamus of a normal brain aged 80 years. The dendritic branches have numerous spines. Golgi staining. Mag. 1200×.

In a morphometric estimation of the mitochondria in dendrites, dendritic spines, and cell body of neurons of the suprachiasmatic nucleus in normal control brains, we concluded that the ellipsoid mitochondria of the spines appear to have an average diameter of 650 ± 250 nm and a mean axial ratio of 1.9 ± 0.2. In addition, the round mitochondria appeared to have a mean diameter of 350 nm. In AD brains, the mitochondria in neurons of

**Figure 6.** Neuron of the SCN of the hypothalamus of a case of AD. The abbreviation of the dendritic arborization and the

poverty of dendritic spines are obvious. Golgi-Nissl staining. Mag. 1200x.

**Figure 5.** Abbreviations of the dendritic arborization is prominent in the neurons of suprachiasmatic nucleus (SCN)

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which is associated with marked decrease in the number of dendritic spines. Golgi staining. Mag. 1200×.

**Figure 5.** Abbreviations of the dendritic arborization is prominent in the neurons of suprachiasmatic nucleus (SCN) which is associated with marked decrease in the number of dendritic spines. Golgi staining. Mag. 1200×.

**Figure 6.** Neuron of the SCN of the hypothalamus of a case of AD. The abbreviation of the dendritic arborization and the poverty of dendritic spines are obvious. Golgi-Nissl staining. Mag. 1200x.

In a morphometric estimation of the mitochondria in dendrites, dendritic spines, and cell body of neurons of the suprachiasmatic nucleus in normal control brains, we concluded that the ellipsoid mitochondria of the spines appear to have an average diameter of 650 ± 250 nm and a mean axial ratio of 1.9 ± 0.2. In addition, the round mitochondria appeared to have a mean diameter of 350 nm. In AD brains, the mitochondria in neurons of

**Figure 4.** Neuron of the SON of the hypothalamus of a normal brain aged 80 years. The dendritic branches have

numerous spines. Golgi staining. Mag. 1200×.

82 Hypothalamus in Health and Diseases

**Figure 3.** Neuron of the SCN of the hypothalamus of a normal brain aged 75 years.

**Figure 7.** Neuron of the SCN of the hypothalamus of a normal brain aged 80 years. The dendritic branches are covered by spines. Golgi staining. Mag. 1200×.

**Figure 8.** Mitochondrial alterations of a dendritic profile of a neuron of SCN of the hypothalamus of a case of AD. Electron micrograph Mag. 124,000×.

the suprachiasmatic nucleus were estimated as having an average diameter of 440 ± 250 nm and a mean axial ratio of 1.7 ± 0.2 (**Table 3**). The round mitochondria appear to have a mean radius of 235 nm. The changes in the morphology of the cristae were also frequent in the mitochondria of hypothalamic neurons in AD, in comparison with normal controls. Morphological alterations of the mitochondria were also seen in a considerable number of astrocytes and pericytes in AD brains.

In a substantial number of neurons of the suprachiasmatic and paraventricular nuclei of the hypothalamus, the Golgi apparatus appeared to be fragmented and atrophic (**Figure 9**). It was noticed, that the atrophy and fragmentation of Golgi apparatus (**Table 4**) and the mitochondrial alterations coexisted frequently with dendritic and spinal pathology in the majority

**Table 3.** Mean diameter (in nm) of mitochondria in neurons of mammillary bodies, based on estimation of 500

**Table 2.** Average dendritic spines per dendritic arbor in SCN and SO neurons, based on measurements of 100 neurons

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of neurons.

mitochondria (p < 0.05).

(p < 0.005).

**Table 2.** Average dendritic spines per dendritic arbor in SCN and SO neurons, based on measurements of 100 neurons (p < 0.005).

the suprachiasmatic nucleus were estimated as having an average diameter of 440 ± 250 nm and a mean axial ratio of 1.7 ± 0.2 (**Table 3**). The round mitochondria appear to have a mean radius of 235 nm. The changes in the morphology of the cristae were also frequent in the mitochondria of hypothalamic neurons in AD, in comparison with normal controls. Morphological alterations of the mitochondria were also seen in a considerable number of

**Figure 8.** Mitochondrial alterations of a dendritic profile of a neuron of SCN of the hypothalamus of a case of AD. Electron

**Figure 7.** Neuron of the SCN of the hypothalamus of a normal brain aged 80 years. The dendritic branches are covered

In a substantial number of neurons of the suprachiasmatic and paraventricular nuclei of the hypothalamus, the Golgi apparatus appeared to be fragmented and atrophic (**Figure 9**). It

astrocytes and pericytes in AD brains.

micrograph Mag. 124,000×.

by spines. Golgi staining. Mag. 1200×.

84 Hypothalamus in Health and Diseases

**Table 3.** Mean diameter (in nm) of mitochondria in neurons of mammillary bodies, based on estimation of 500 mitochondria (p < 0.05).

was noticed, that the atrophy and fragmentation of Golgi apparatus (**Table 4**) and the mitochondrial alterations coexisted frequently with dendritic and spinal pathology in the majority of neurons.

the early stages of AD. The suprachiasmatic nucleus seems to be more seriously affected than the others in aging [76] and degenerative disorders. In previous studies, it was clearly revealed that the total cell population of the suprachiasmatic nucleus is decreased both in aging and in AD [76], in which the hypothalamic dysfunction is closely related to sleep disturbances [77]. The hypothalamic nuclei seems to be involved in those neurodegenerative alterations, which would progressively result in AD. In addition, the comparison of the morphological and morphometric alterations of the dendrites in the hypothalamic nuclei with those observed in the cortex of the brain hemispheres and the cerebellum disclosed that the alterations in the hypothalamus were rather modest, in correlation with those of the acoustic and visual cortices, the

The Hypothalamus in Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.81475 87

The fact that the hypothalamus is the principal subcortical center for the homeostatic and autonomic processes may explain the reason that the supraoptic and the periventricular nuclei,

However, the suprachiasmatic nucleus demonstrated more severe dendritic alterations and synaptic loss than the supraoptic and paraventricular ones, a fact which might explain the phenomenon of desynchronization of circadian rhythms in the majority of the patients, who suffer from AD [82] and cognitive decline [83] in the spectrum of other degenerative brain disorders [84], given that the suprachiasmatic nucleus is indispensable for the generation and synchronization of circadian rhythms in man [85, 86]. It is reported that changes of the circadian rhythm (CR), arterial blood pressure, and body temperature may occur in AD patients [87] especially during the night time [88–90]. Changes also of the melatonin levels are not an unusual phenomenon in advanced senility and AD [91–93]. Sundown syndrome, on the other hand, frequently associated with increased motor activity, is a rather common phenomenon in advanced AD cases [93].

In the majority of neurons of hypothalamic nuclei, mitochondrial alterations were prominent in the cell body as well as in dendrites and synaptic components. As the mitochondria play an essential role in the energy supply of the cell, as ATP-generating organelles, their role is of utmost importance in the alteration of reduction-oxidation potential of the cell, in the free radical formation and scavenging, in the intracellular calcium control and the eventual activation of apoptotic chain [94–96]. Normally, the number of dendritic, axonic, and synaptic mitochondria is very high, especially in preand post-synaptic components, since they are the major energy contributor for the synaptic activity.

Mitochondrial dysfunction might induce Aβ peptide neurotoxicity, whereas enhancing mitochondrial proteostasis may reduce amyloid-β proteotoxicity [97]. In addition, impaired mitochondrial biogenesis contributes to mitochondrial dysfunction [98], which is directly associated with oxidative stress, activating furthermore the pathogenic cascade of AD [99–101]. Mitochondrial motility and accumulation are related to the functional state of the neuron, since mitochondria are transported to regions where the necessity for energy is particularly high, as it occurs in the dendritic and axonal profiles and the synapses [102–104]. The shape and size of mitochondria are not stable, since they undergo continual fission and fusion, which are necessary both for

Recent studies reported increased mitochondrial fission and decreased fusion, due to increased Aβ peptide interaction with the mitochondrial fission protein Drp 1, inducing increased mitochondrial fragmentation, impaired axonal transport of mitochondria, and

the survival of the cell and the harmonious adaptation to changing conditions.

among others, reserve substantial synaptic density, even at the advanced stages of AD.

prefrontal areas of the brain, and the cerebellar cortex [78–81].

**Figure 9.** Alteration of Golgi apparatus of a neuron of the SCN of the hypothalamus of a case of AD. Electron micrograph. Mag. 124,000×.

**Table 4.** The volume of Golgi apparatus in nm3 based on measurements of 100 neurons of SCN (p < 0.005).

#### **4. Discussion**

Hypothalamus is a crucial brain structure for the regulation and control of essential homeostatic functions, including the circadian rhythms (CRs) and the sleep-wake cycle. In Alzheimer's disease and other neurodegenerative disorders [74–76], several hypothalamic nuclei are affected. It seems that the hypothalamic nuclei are not involved simultaneously at the early stages of AD. The suprachiasmatic nucleus seems to be more seriously affected than the others in aging [76] and degenerative disorders. In previous studies, it was clearly revealed that the total cell population of the suprachiasmatic nucleus is decreased both in aging and in AD [76], in which the hypothalamic dysfunction is closely related to sleep disturbances [77].

The hypothalamic nuclei seems to be involved in those neurodegenerative alterations, which would progressively result in AD. In addition, the comparison of the morphological and morphometric alterations of the dendrites in the hypothalamic nuclei with those observed in the cortex of the brain hemispheres and the cerebellum disclosed that the alterations in the hypothalamus were rather modest, in correlation with those of the acoustic and visual cortices, the prefrontal areas of the brain, and the cerebellar cortex [78–81].

The fact that the hypothalamus is the principal subcortical center for the homeostatic and autonomic processes may explain the reason that the supraoptic and the periventricular nuclei, among others, reserve substantial synaptic density, even at the advanced stages of AD.

**Figure 9.** Alteration of Golgi apparatus of a neuron of the SCN of the hypothalamus of a case of AD. Electron micrograph.

Hypothalamus is a crucial brain structure for the regulation and control of essential homeostatic functions, including the circadian rhythms (CRs) and the sleep-wake cycle. In Alzheimer's disease and other neurodegenerative disorders [74–76], several hypothalamic nuclei are affected. It seems that the hypothalamic nuclei are not involved simultaneously at

based on measurements of 100 neurons of SCN (p < 0.005).

Mag. 124,000×.

86 Hypothalamus in Health and Diseases

**4. Discussion**

**Table 4.** The volume of Golgi apparatus in nm3

However, the suprachiasmatic nucleus demonstrated more severe dendritic alterations and synaptic loss than the supraoptic and paraventricular ones, a fact which might explain the phenomenon of desynchronization of circadian rhythms in the majority of the patients, who suffer from AD [82] and cognitive decline [83] in the spectrum of other degenerative brain disorders [84], given that the suprachiasmatic nucleus is indispensable for the generation and synchronization of circadian rhythms in man [85, 86]. It is reported that changes of the circadian rhythm (CR), arterial blood pressure, and body temperature may occur in AD patients [87] especially during the night time [88–90]. Changes also of the melatonin levels are not an unusual phenomenon in advanced senility and AD [91–93]. Sundown syndrome, on the other hand, frequently associated with increased motor activity, is a rather common phenomenon in advanced AD cases [93].

In the majority of neurons of hypothalamic nuclei, mitochondrial alterations were prominent in the cell body as well as in dendrites and synaptic components. As the mitochondria play an essential role in the energy supply of the cell, as ATP-generating organelles, their role is of utmost importance in the alteration of reduction-oxidation potential of the cell, in the free radical formation and scavenging, in the intracellular calcium control and the eventual activation of apoptotic chain [94–96]. Normally, the number of dendritic, axonic, and synaptic mitochondria is very high, especially in preand post-synaptic components, since they are the major energy contributor for the synaptic activity.

Mitochondrial dysfunction might induce Aβ peptide neurotoxicity, whereas enhancing mitochondrial proteostasis may reduce amyloid-β proteotoxicity [97]. In addition, impaired mitochondrial biogenesis contributes to mitochondrial dysfunction [98], which is directly associated with oxidative stress, activating furthermore the pathogenic cascade of AD [99–101]. Mitochondrial motility and accumulation are related to the functional state of the neuron, since mitochondria are transported to regions where the necessity for energy is particularly high, as it occurs in the dendritic and axonal profiles and the synapses [102–104]. The shape and size of mitochondria are not stable, since they undergo continual fission and fusion, which are necessary both for the survival of the cell and the harmonious adaptation to changing conditions.

Recent studies reported increased mitochondrial fission and decreased fusion, due to increased Aβ peptide interaction with the mitochondrial fission protein Drp 1, inducing increased mitochondrial fragmentation, impaired axonal transport of mitochondria, and synaptic degeneration in AD [99]. The consequence of the dynamic fusion and fission processes is the eventual mitophagy of the damaged mitochondria.

Mitochondrial alterations and fragmentation of Golgi complex are observed by electron microscopy in a substantial number of neurons and astrocytes in the hypothalamic nuclei. The hypothalamic pathology may be related to instability of autonomic regulation and homeo-

, Demetrios Mitilineos1

1 Laboratory of Neuropathology and Electron Microscopy, Department of Neurology,

[1] Alzheimer A. Über eine eigenartige Erkrankung der Hirnrinde. Allgemeine Zeitschrift

[2] Blessed G, Tomlinson BE, Roth M. The association between quantitative measures of dementia and of senile change in the cerebral grey matter of elderly subjects. The British

[3] Reitz C, Mayeux R. Alzheimer disease: Epidemiology, diagnostic criteria, risk factors and

, Ioannis S. Baloyannis1

The Hypothalamus in Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.81475 89

static disequilibrium, which are gradually established in Alzheimer's disease.

**Conflict of interest**

No conflict of interest.

**Author details**

**References**

and Vassiliki G. Costa1,2

**Nomenclature and abbreviations**

PVN paraventricular nucleus

Stavros J. Baloyannis1,2\*, Ioannis Mavroudis1

Aristotelian University, Thessaloniki, Greece

für Psychiatrie. 1907;**64**:146-148

Journal of Psychiatry. 1968;**114**:797-811

\*Address all correspondence to: sibh844@otenet.gr

2 Institute for Research on Alzheimer's Disease, Iraklion, Greece

biomarkers. Biochemical Pharmacology. 2014;**88**:640-651

SCN superchiasmatic nucleus of the hypothalamus

SON supraoptic nucleus of the hypothalamus

HPA hypothalamic-pituitary-adrenal pathway

AD Alzheimer's disease

A prominent decrease of the size of the mitochondria is observed in aging-related neurodegenerative diseases [95, 96], as well as at the early stages of AD, prior to the onset of a noticeable cognitive dysfunction [105]. Normally, a limited number of dendritic spines contain small and round mitochondria, which are increased in number in the dendritic profiles during the synaptogenesis and hormonal instability [102, 104]. It is important to underline that mitochondrial alterations are mostly associated with synaptic loss in AD patients, due to impairment of mitochondrial energy production [106], seen even before the typical generation of the neuritic plaques and tau pathology [105, 107].

The morphological alteration of the mitochondria, seen in the hypothalamic nuclei in early cases of Alzheimer's disease, pleads in favor of a generalized mitochondrial dysfunction in AD, which may be associated with the dendritic pathology, the tremendous loss of spines, and the marked synaptic alterations [108–110].

The density of the spines on the dendritic branches of a considerable number of neurons of the suprachiasmatic nucleus was decreased. The loss of the dendritic spines causes substantial impairment in neuronal communication and also induces reasonable dysfunction of the neuronal circuits in AD. Previous observations revealed that the loss of dendritic spines coincides with the morphological alterations of the mitochondria and the fragmentation of the cisternae of Golgi apparatus [25, 102, 109, 110]. In an experimental mouse model of Aβ peptide deposition, it was revealed that nonfibrillar Aβ peptide may exert toxicity on the spines, resulting in dramatic decrease of spine density [108, 111].

The role of the hypothalamus in the harmonization of circadian rhythms is crucial for the maintenance of energy homeostasis [25]. The feeding behavior [111–113] and the thermoregulation of the body become gradually unstable during the clinical course of AD [114–116], a fact which was also noticed in experimental models of AD [117] as well as in the behavioral variant of fronto-temporal dementia [118].

In conclusion, the hypothalamic nuclei are involved in AD, inducing autonomic dysfunction and homeostatic disequilibrium, phenomena which are clearly noticeable at the advanced stages of AD.
