AVP-eGFP was significantly upregulated by hypovolemia in the parvocellular division of the paraventricular nucleus in the transgenic rats

Arginine vasopressin (AVP) is produced in the paraventricular (PVN) and supraoptic nuclei (SON). Peripheral AVP, which is secreted from the posterior pituitary, is produced in the magnocellular division of the PVN (mPVN) and SON. In addition, AVP is produced in the parvocellular division of the PVN (pPVN), where corticotrophin releasing factor (CRF) is synthesized. These peptides synergistically modulate the hypothalamic-pituitary-adrenal (HPA) axis. Previous studies have revealed that the HPA axis was activated by the hypovolemia. However, the detailed dynamics of AVP in the pPVN under hypovolemic state has not been elucidated. Here, we evaluated the effects of hypovolemia and hyperosmolality on the hypothalamus, using AVP-enhanced green fluorescent protein (eGFP) transgenic rats. Polyethylene glycol (PEG) or 3% hypertonic saline (HTN) was intraperitoneally administered in order to develop hypovolemia or hyperosmolality. AVP-eGFP intensity was robustly upregulated at 3 and 6 h after intraperitoneal (i.p.) administration of PEG or HTN in the mPVN. While in the pPVN, eGFP intensity was significantly increased at 6 h after i.p. administration of PEG with significant induction of Fos-immunoreactive (-ir) neurons. Consistently, eGFP mRNA, AVP hnRNA, and CRF mRNA in the pPVN and plasma AVP and corticosterone were significantly increased at 6 h after i.p. administration of PEG. The results suggest that AVP and CRF syntheses in the pPVN were activated by hypovolemia, resulting in the activation of the HPA axis.

The influence of melatonin on stress-induced ultramicroscopic changes of the neurons of the supraoptic nuclei of the rat hypothalamus

The aim: to study the effect of melatonin on the ultrastructural state of the supraoptic nuclei of the hypothalamus of rats under immobilization stress.Materials and methods. The experiments were performed on non-linear male white rats weighing 200-220 g. The animals were divided into 3 study series, in each of which the biomaterial was collected at 2 p.m. and at 2 a.m. using electron microscopic method. Long immobilization stress was simulated by keeping rats in special plastic penal cages for 6 hours daily for 7 consecutive days. Melatonin (Sigma, USA, 99.5% purification degree) at a dose of 0.5 mg/kg, in 1.0 ml of solvent (0.9% ethanol solution on physiologic saline) was injected daily, intraperitoneally.Results. When the animals were kept under the standard light regime, the ultrastructural organization of the hypothalamic nuclei at 2 p.m. indicated their low functional activity in comparison with the studies carried out at 2 a.m. Prolonged exposure of rats to immobilization stress was reflected in a significant rearrangement of the ultrastructural organization of supraoptic nuclei of the hypothalamus. The established changes can be considered as a manifestation of neurosecretory activity suppression, a decrease in neurosecretase production by hypothalamic neurons. Melatonin injections against the background of immobilization stress resulted in relative normalization of ultrastructural state of neurons of supraoptic nuclei of the hypothalamus of animals. In particular, studies at 2 a.m. revealed light neurosecretory cells containing a large nucleus, it was pyknotically altered. Karyolema invaginations, euchromatin dominance in the nucleus were observed. Heterogeneous changes were observed on the part of mitochondria. Enlarged tubules of granular endoplasmic reticulum were seen. At the same time, a small number of ribosomes and few hormonal granules were noticeable in neuroplasm. The mentioned picture of neurosecretory cells reflects a relative improvement in their electron microscopic state, which is evidenced by the appearance of neurosecretory granules. However, the ultrastructure of other organelles of the studied neurons indicates a depleted state caused by prolonged immobilization.Conclusions. 1. In animals under standard photoperiod conditions, the structural organization of supraoptic neurons of the hypothalamic nuclei during the nighttime of the experiment reflects the intensity of intracellular synthesizing processes (at 2 a.m.). A decrease in the activity of the structures under study is noted during the daytime. 2. Under immobilization stress, the ultrastructural organization of the above neurons indicates a pronounced disturbance of reactive nature with the signs of decreased functional ability of the structures and the phenomena of edema and destruction during the period of observation. 3. Melatonin injections against the background of immobilization stress led to a relative improvement in the ultrastructural state of the animals’ hypothalamic nuclei neurons, which is evidenced by the appearance of neurosecretory granules. However, the ultrastructure of other organelles of the studied neurons indicated a depleted state caused by prolonged immobilization.

Morphometric analysis of supraoptic neurons of the rat hypothalamic nuclei under conditions of prolonged illumination

The article reviews the results of studies of the morphofunctional state of neurons of the supraoptic nuclei of the rat hypothalamus under conditions of different duration of light regime. Under standard light regime in rats, a diurnal rhythm of morphofunctional activity of supraoptic nucleus neurons with maximum activity during daytime (before 2 p.m.) is recorded. In animals subjected to prolonged light exposure, more pronounced changes in the morphofunctional state of the supraoptic neurons of the hypothalamus at 2 a.m. than at 2 p.m. were established. Thus, the neuronal nucleus area was 94.08 ± 9.55 μm2 and was significantly greater than that in intact animals. The nucleo-cytoplasmic ratio of supraoptic hypothalamic neuron at 2 a.m. was lower than that in intact animals due to a decrease in specific nucleus volume. In comparison with the day period (2 p.m.), before 2 a.m. there was revealed a decrease of the neuron body area of supraoptic nuclei of hypothalamus due to possible decrease of the area of nucleus and nucleolus of cells. This was the reason for the increase in the nucleo-cytoplasmic ratio in the neurons under observation at night, which was 2.51 ± 0.023 units. Constant light regime did not cause inversion of the rhythm of morphofunctional activity of the neurons under study, the maximum values, as in intact animals, occurred in the daytime observation period.

The Same Magnocellular Neurons Send Axon Collaterals to the Posterior Pituitary and Retina or to the Posterior Pituitary and Autonomic Preganglionic Centers of the Eye in Rats

In rats, some parvocellular paraventricular neurons project to spinal autonomic centers. Using the virus tracing technique, we have demonstrated that some magnocellular paraventricular neurons, but not supraoptic neurons, also project to autonomic preganglionic centers of the mammary gland, gingiva, or lip. A part of these neurons has shown oxytocin immunoreactivity. In the present experiment, we have examined whether the same magnocellular neuron that sends fibers to the retina or autonomic preganglionic centers of the eye also projects to the posterior pituitary. Double neurotropic viral labeling and oxytocin immunohistochemistry were used. After inoculation of the posterior pituitary and the eye with viruses, spreading in a retrograde direction and expressing different fluorescence proteins, we looked for double-labeled neurons in paraventricular and supraoptic nuclei. Double-labeled neurons were observed in non-sympathectomized and cervical-sympathectomized animals. Some double-labeled neurons contained oxytocin. After the optic nerve was cut, the labeling did not appear in the supraoptic nucleus; however, it could still be observed in the paraventricular nucleus. In the paraventricular nucleus, the double-labeled cells may be the origin of centrifugal visual fibers or autonomic premotor neurons. In the supraoptic nucleus, all double-labeled neurons are cells of origin of centrifugal visual fibers.

НЕЙРОДЕГЕНЕРАТИВНЫЕ ИЗМЕНЕНИЯ В ВЕСТИБУЛЯРНЫХ НЕЙРОНАХ ПОСЛЕ ОДНОСТОРОННЕЙ ЛАБИРИНТЭКТОМИИ (UL) И ИХ ЗАЩИТА ГИПОТАЛАМИЧЕСКИМ ПРОЛИН БОГАТЫМ ПЕПТИДОМ (PRP) И ЗМЕИНЫМ ЯДОМ NAJA NAJA OXIANA (NOX) / NEURODEGENERATIVE CHANGES IN VESTIBULAR NEURONS AFTER UNILATERAL LABYRINTHECTOMY (UL) AND THEIR PROTECTION WITH HYPOTHALAMIC PROLINE RICH PEPTIDE (PRP) AND SNAKE VENOM NAJA NAJA OXIANA (NOX)

Мы протестировали реакции нейронов ядра Дейтерса на двустороннюю высокочастотную стимуляцию паравентрикулярных и супраоптических ядер гипоталамуса (PVN & SON) в норме и после односторонней лабиринтэктомии (UL). Анализ спайковой активности проводился с помощью on-line выборки и специальной программы. Комплексные усредненные гистограммы времени и частоты перисобытий показывают усиление тормозных и возбуждающих реакций нейронов Дейтерса на ранней стадии вестибулярной компенсации после инъекции богатого пролином пептида (PRP-1) и яда кобры Naja Naja Oxiana (NOX), достигая норме по окончании испытаний. При гистохимическом исследовании обнаружено изменение активности Са2+-зависимой кислой фосфатазы (КФ) в нейронах. Было показано, что у UL животных полное исчезновение или задержка обесцвечивания нейронов Дейтерса приводит к нейродегенеративному паттерну в виде клеточного «оттенка». Активность КФ после UL и инъекции PRP-1 вызывает более эффективное восстановление нейронов по сравнению с событиями, наблюдаемыми после введения NOX. Наблюдения за поведением в «открытом поле» показывают, что PRP-1 и NOX являются протекторами, которые могут успешно восстанавливать нарушенные вестибулярные функции. / We tested the reactions of Deiters’ nucleus neurons to bilateral high frequency stimulation of hypothalamic paraventricualar and supraoptic nuclei (PVN & SON) in norm and following unilateral labyrinthectomy (UL). The analysis of spike activity was carried out by mean of on-line selection and special program. The complex averaged perievent time and frequency histograms shows the increase of inhibitory and excitatory reactions of Deiters’ neurons at early stage of vestibular compensation following proline-rich peptide (PRP-1) and cobra venom Naja Naja Oxiana (NOX) injection, reaching the norm at the end of tests. In histochemical study the changes in Ca2+-dependent acidic phosphatase (AP) activity in neurons was discovered. It was shown that in UL animals the total disappearance or delay of decolorizing of Deiters’ neurons lead to neurodegenerative pattern as cellular “shade”. AP activity after UL and PRP-1 injection exerts more effective recovery of neurons in comparison with events, observed after the administration of NOX. The behavioral observations in “open fieald” indicate that PRP-1 and NOX are protectors, which may successfully recover the disturbed vestibular functions.

SUBMICROSCOPIC TRANSFORMATIONS OF THE HYPOTHALAMUS STRUCTURE UNDER CONDITIONS OF 24-HOUR LIGHTING

Light information that is perceived by the retinal photoreceptors is transmitted along the retinohypothalamic pathway (the retinal ganglion cells present in it) and supraoptic fibers, suprachiasmatic, paraventricular, arcuate nuclei of the hypothalamus, through the trunk of the upper thoracic region and lateral intermedial nuclei of the spinal cord, sym- pathic neurons of the superior cervical ganglion in the pineal gland. In the dark, signals from the suprachiasmatic nucleus enhance the synthesis and release of norepinephrine from sympathetic endings. The experiments were carried out on 40 sexually mature male outbred white rats weighing 0.15-0.18 kg. The animals were kept in cages at a constant temperature, air humidity and free access to water and food. Experimental animals were divided into two series, in each of which the biomaterial was taken at 14.00 and 02.00 h. The selected timing of the experiment is due to the different functional activity of the pineal gland in the indicated time periods of the day. Submicroscopic studies of the supraoptic nuclei of the hypothalamus of intact animals at 14:00 showed that the majority of neurosecretory cells are round-oval in shape with rare invaginations and nuclei of irregular shape with shallow invaginations of the karyolem. The karyoplasm contains lumps of chromatin and a dense osmiophilic nucleolus. Neuroplasm occupies a small volume, in it the tubules of the granular endoplasmic reticulum are tightly packed with a small lumen, where there are many ribosomes and polysomes, as well as a small amount of evenly distributed granules. In the Golgi complex, secretory granules of different sizes are formed. Mitochondria, small with a dense matrix, contain few cristae. Some of them are in an energetically stressed state, are able to hypertrophy and partially lose their cristae or die. The ultramicroscopic organization of neurosecretory cells of the supraoptic nuclei of the anterior hypothalamus of rats was investigated. Under the standard illumination mode (12.00C: 12.00T), the ultrastructure of neurons indicates a decrease in their functional activity during the light period of the day and growth - in the dark period of the day. Light stress (24.00C: 00T) leads to significant desynchronosis and causes destructive changes in the components of the structures under study, more pronounced at 02.00 h.

NEURODESTRUCTION OF HYPOTHALAMIC NUCLEI IN BRAIN INJURY. EFFECT OF CARBACETAM

Relevance. A key role in the pathogenesis of brain injury (BI) is played by destructive changes in the neural tissue of the brain, which consist in damage to neurons and glial cells. To date, various drugs are being intensively developed and studied, which are considered in the perspective of correction and restoration of the functional state of the brain. These substances include the neuroprotector carbacetam, an modulator of the GABA-benzodiazepine receptor complex, a derivative of the alkaloid β-carboline. Objectie. To investigate the effect of carbacetam on neurodestruction processes in the paraventricular and supraoptic nuclei of the hypothalamus in experimental BI. Material and methods. The study was carried out on 20 white non-native male rats weighing 200±10 g. To simulate the BI, rats were subjected to one stroke along the cranial vault with a free-fall load according to the V.N. Yelskyy and S.V. Ziablitsev method (2008). The energy of impact was 0.52 J, the lethality for the first 5 days after injury was 84%. In the control group (n=10) 1 ml of saline was injected intraperitoneally once daily for 10 days after injury. Animals of the experimental group (n=10) received intraperitoneally injections of carbacetam at a dose of 5 mg/kg in 1 ml of saline according to the same scheme. After the experiment was over, the animals were decapitated with the removal of the brain, from which histological preparations were made with a microtome after appropriate histological treatment. Some sections were stained with hematoxylin and eosin, others were immunohistochemically reacted with antibodies against neuronmarkers proteins NSE, S-100 and GFAP. Results. Carbacetam influenced the decrease of degenerative processes in the nervous tissue of the paraventricular and supraoptic nuclei of the hypothalamus. Neurons of animals with BI that received carbacetam, were characterized by the restoration of normal morphological features in contrast to rats not receiving the drug. Immunohistochemical study of brain neuromarkers confirmed the restoration of the functions of neurons and astrocytes in the investigated parts of the rat's hypothalamus after the administration of carbacetam. There was a decrease in the expression level of glial markers GFAP and S-100, which illustrated the decrease in degenerative changes in the nervous tissue. While the expression level of the neuron marker NSE grew, this demonstrated the high metabolic activity of nerve cells. Changes in the expression of markers of neurons and glia indicated a restoration of normal neuronal activity under the action of carbacetam. Conclusion. Further investigation of the effects of carbacetam seems promising in terms of the restoration of neuronal function at BI.