The comparisons on total RNA from different source-original neurons applied in LMPC.

Objective To compare the quality and quantity of total RNA from different source-original neurons applied in LMPC technique. Methods (1) Aglient 2100 bioanalyzer and RT-PCR were used to check the concentration and fragmentation of total RNA from unfixed, temporal fixed and fixed 12 h hypothalamus sections; (2) Different neurons of PVN and SON were collected by LMPC, CRH, TRH, AVP, OT mRNA level were measured by RT-PCR; (3) Labeled neurons by injecting CTB into stomach and non-labeled neurons in DMV collected by LMPC were checked for house keeping genes by RT-PCR. Results (1) Unfixed section had higher concentration and better quality of total RNA compared with fixed sections applied in LMPC; relative short amplicons such as GAPDH, NSE, MCH and MC4R were successfully obtained from fixed and unfixed and long amplicon of GR can only be obtained from unfixed material; (2) In mangocellular PVN and SON the expressions of AVP and OT were more special than those in the parvocellular PVN. Oppositely, the expressions of CRH, TRH in the parvocellular were more special than the other two; (3) The expressions of house keeping genes had no significant difference between labeled and non-labeled DMV neurons. Conclusion The quality and quantity of total RNA from unfixed brain tissues were better than fixed tissues applied in LMPC and the CTB tracer which may differentiate neurons had no significant effect on physiology of the neurons applied in LMPC. The results showed that the LMPC technique is suitable for the qualitative and quantitative study on individual neurons at mRNA level.

High fat diet induced hepatic insulin resistance is not related to changes in hypothalamic mRNA expression of NPY, AgRP, POMC and CART in mice.

The hypothalamic circuitry, apart from its impact on food intake, modulates insulin sensitivity to adapt metabolic conditions in the face of environmental fluctuations in nutrient availability. The purpose of the present study was to investigate the effects of 2 weeks high fat feeding in wildtype mice on (1) insulin sensitivity and triglyceride accumulation in liver and muscle in relation to (2) mRNA expression levels of Neuropeptide Y (NPY), Agouti-related protein (AgRP), pro-opiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus. Two weeks of high fat feeding induced hepatic insulin resistance in the presence of increased hepatic triglyceride accumulation. In muscle, however, 2 weeks of high fat feeding did not result in changes in insulin sensitivity or in triglyceride content. mRNA expression levels of NPY, AgRP, POMC, and CART in the hypothalamus were not different between the groups. This study shows that 2 weeks of high fat feeding in mice does not affect mRNA expression levels of NPY, AgRP, POMC or CART, in the whole hypothalamus, despite induction of hepatic, but not peripheral, insulin resistance. Therefore, a major physiological role of these neuroendocrine factors in the induction of hepatic insulin resistance during a high fat diet seems less likely.

The biological clock tunes the organs of the body: timing by hormones and the autonomic nervous system.

The biological clock, the suprachiasmatic nucleus (SCN), is essential for our daily well-being. It prepares us for the upcoming period of activity by an anticipatory rise in heart rate, glucose and cortisol. At the same time the 'hormone of the darkness', melatonin, decreases. Thus, the time-of-day message penetrates into all tissues, interestingly not only by means of hormones but also by a direct neuronal influence of the SCN on the organs of the body. The axis between the SCN and the paraventricular nucleus of the hypothalamus (PVN) is crucial for the organization/synchronization of the neuroendocrine and autonomic nervous system with the time of day. This SCN-neuroendocrine PVN axis takes care of a timely hormonal secretion. At the same time, the SCN-autonomic PVN axis fine-tunes the organs by means of the autonomic nervous system for the reception of these hormones. Finally, the similar organization of the projections of the human SCN as compared with that in the rodent brain suggests that these basic principles of neuroendocrine autonomic interaction may also be true in the human. The physiological data collected in humans thus far seem to support this hypothesis, while pathological changes in the SCN of humans suffering from depression or hypertension indicate a role for the SCN in the etiology of these diseases.

Retrograde degeneration of thalamic neurons in the mediodorsal nucleus after neonatal and adult aspiration lesions of the medial prefrontal cortex in the rat. Implications for mechanisms of functional recovery.

The behavioural consequences of neonatal lesions of the frontal cortex are limited as compared with similar lesions performed in adulthood. The present study has investigated, using unbiased quantitative methods with randomized systematic sampling, the total neuronal cell numbers in the mediodorsal nucleus of the thalamus after aspiration lesions of the medial prefrontal cortex performed in neonatal and in adult rats. It was found that the reduction in total cell numbers after neonatal prefrontal cortex lesions was similar to that found after adult cortex lesions. In neonatally lesioned animals the neuronal cell density was significantly increased by 13%, whereas in adult lesioned animals it was unchanged. On the other hand, the volume of the mediodorsal nucleus was reduced by 27% in neonatally, and 20% in adult lesioned animals. Total neuronal cell number of the mediodorsal nucleus was significantly decreased in neonatally as well as in adult lesioned rats, by 14% and 21%, respectively. These findings are discussed in the light of the previously proposed role of the thalamus as a neural substrate of functional sparing after neonatal cortical lesions.

Developmental pattern of NADPH-diaphorase activity and nitric oxide-stimulated cGMP immunoreactivity in the frontal rat cortex and its role in functional recovery from aspiration lesions.

The present study was undertaken to (1) explore the cortical nitric oxide (NO)-system during postnatal development and (2) to see whether or not the NO-system reacts differentially after neonatal and adult lesions of the medial prefrontal cortex. Three aspects of the NO-system were studied, i.e., NADPH-diaphorase (NADPH-d) activity, sodium nitroprusside (SNP)- and N-methyl-D-aspartate (NMDA)-stimulated cGMP-immunoreactivity (cGMP-IR). It was shown that: (1) the development of NADPH-d activity containing cells is continued in the period from P6 until P21; (2) during the same period, large developmental changes take place in basal, and SNP- or NMDA-stimulated cGMP-IR in the cortex. These changes are regionally specific and follow the general cortical developmental pattern; and (3) aspiration lesions do not induce major changes in the distribution of NADPH-d activity or cGMP-IR, either basal, SNP- or NMDA-stimulated.

Laminar distribution of GABAA receptor alpha 1, beta 2, and gamma 2 subunit mRNAs in the granular and agranular frontal cortex of the rat during pre- and postnatal development.

The expression of mRNAs encoding the three GABAA receptor subunits that are associated with the most abundant benzodiazepine-sensitive GABAA receptor in adult cortex, that is, the alpha 1, beta 2, and gamma 2 subunits, was studied in rat cortex during pre- and postnatal development by means of in situ hybridization, gamma 2 and beta 2 mRNAs become detectable in neocortex at gestational day 16 (GD16), alpha 1 at GD18. gamma 2 mRNA exhibits the highest level of expression at early ages, while alpha 1 mRNA levels are low. beta 2 mRNA rises steeply during the last days of gestation. Around birth, it shows the highest expression of the three subunits studied in cortex, and increases further until postnatal day 15 (PD15). The expression of alpha 1 subunit mRNA also increases markedly shortly before birth and accelerates between PD8 and PD15, when it reaches higher levels than the other two subunits. Following the initial high expression, gamma 2 mRNA increases gradually and slowly until PD25. During prenatal development, highest expression of all three subunit mRNAs is found in the upper layers of cortex, that is, cortical plate and marginal zone. The subplate layer does not start to express GABAA receptor subunit mRNAs until GD18. At birth, all developing layers of the cortex express mRNAs for the three subunits, except the marginal zone. Highest levels are found in the upper part of the cortical plate. At the end of the first postnatal week (PD8), the laminar distribution of mRNA expression in neocortex becomes more differentiated. For all three subunit mRNAs, highest expression is then observed in neuron-like cells in layer IV in the granular areas, and over layers III and upper V in agranular areas. Subsequently, between PD8 and PD25, increasing levels of expression are observed over the pyramidal cell layer V. This regionally differentiated, developmental pattern suggests a close relationship between development of GABAA receptor subunits, ingrowth of thalamocortical projections, and maturation of neocortical circuitry.

Lesion induced expression of low-affinity NGF-binding protein (p75) immunoreactivity after neonatal and adult aspiration lesions of the rat dorsomedial prefrontal cortex.

The present study was performed in order to examine whether or not NGF-mediated processes could be involved in the sparing of function observed after neonatal prefrontal cortex lesions. After unilateral neonatal aspiration lesions of the dorsomedial prefrontal cortex, fibers immunoreactive for the low-affinity NGF-binding protein (p75) with a deviant morphology were observed in the severed hemisphere only. The morphology of these fibers was characterized by their large caliber, their large, often bulbous varicosities, and their curly appearance. These fibers were present as soon as 24 h after the operation. Between 3 and 5 days after the operation, the greatest abundance of these fibers was found in the ventrorostral areas of the forebrain and along the pathways of cortical projections of the cholinergic cell groups. After 7 days, such fibers were no longer observed. After comparable lesions in adult animals, a similar type of fiber was observed in the lesioned hemisphere. However, in these cases a response comparable to that observed in the neonatal animals was not observed until 5 days after the operation, with fewer fibers. Furthermore, in contrast to what was observed after neonatal lesions, in adult animals no indications of retrograde transport of p75 immunoreactive material towards the cholinergic cells of the basal forebrain nuclei were found. From these findings it was concluded that the prompt upregulation of p75 expression in neonatal animals may contribute to the survival of the cholinergic cells of the basal forebrain, and may therefore be involved in the restoration of function of the medial prefrontal cortex.

Effects of neonatal mediodorsal thalamic lesions on structure and function of the rat prefrontal cortex.

The morphological and behavioral effects of neonatal electrothermal lesions of the mediodorsal thalamus on the development of the prefrontal cortex were studied. Lesions of the mediodorsal nucleus (MDT), inflicted on the day of birth, caused no significant changes in prefrontal architecture on day 35. On the other hand, a significant decrease in cortical width (4.7-7.7%) was observed at some places within the lateral and supragenual parts of the prefrontal cortex. However, these local decreases in cortical width were not reflected by a significant decrease of the total volume of the particular prefrontal subareas. In adulthood, rats with neonatal MDT lesions were exposed to an operant delayed alternation task, which is known to depend upon the integrity of the prefrontal cortex, in order to investigate the behavioral consequences of the lesions for prefrontal functioning. The lesions did not impair the rats abilities to learn the spatial delayed alternation task. Neonatally lesioned and control rats scored equally. Given the relatively mild effects of MDT lesioning, thalamic fibers do not seem to play a crucial role, at least not during the postnatal period of prefrontal cortical development. It is discussed whether or not this is a specific characteristic of agranular association cortex, in which the termination of thalamic and cortical afferents overlap in layer III.

Oligodendroglioma: the Rotterdam-Dijkzigt experience.

In a retrospective study of 82 cases of oligodendroglioma, the influences of tumor size, site, and grade, the age of the patient, the extent of surgical excision, and the effect of additional radiation therapy on the clinical course, as well as their dependencies, were investigated. Tumor grade and tumor site significantly affected the survival rates of the patients. When tumor site was kept constant by distinguishing between frontal and nonfrontal localized oligodendrogliomas, the grading system still showed discriminating power. Conversely, when controlled for grade, the frontal site appeared to be favorable for prognosis. Age was only faintly correlated with survival, with younger patients tending to survive longer. This was compatible with the finding that, in young patients, more frontal localized tumors were found and that older patients had oligodendrogliomas with higher grades. Tumor volumes did not correlate with survival. No correlation between tumor volume and tumor grade was found. Patients who had undergone a decompression tended toward longer survival, although this trend did not reach significance. No beneficial effect of radiation therapy on the survival rate was demonstrable.

Expression of p53 in oligodendrogliomas.

The expression of the nuclear protein p53 in oligodendrogliomas was investigated by immunohistochemistry, using a monoclonal anti-p53 antibody (DO-7) on formalin-fixed, paraffin-embedded material in 84 histologically verified cases, and compared with the histopathological grade and survival. p53-immunoreactive cells were found in 75 per cent of the samples acquired at the first biopsy. The p53 labelling index was not related to the degree of nuclear anaplasia. Tumour cases with more than 75 per cent p53 immunostained cells had a rapidly fatal clinical course. However, no significant correlation was found between p53 labelling index and tumour grade, mitotic index, or ploidy status. In most tumour recurrences (n = 25), the p53 labelling index increased or remained at the level of the first biopsy. In five cases (6 per cent), p53 was absent in the first sample as well as in the recurrence. Irrespective of the underlying aberration of either the gene or the metabolic pathway of p53, it is concluded that a high percentage (i.e., more than 75 per cent) of p53-immunolabelled cells is predictive of an unfavourable clinical course, while a percentage lower than 75 per cent immunoreactive cells does not exclude a rapid fatal outcome.

Ineffectiveness of GM1 and ORG2766 on behavioural recovery after prefrontal cortical lesions in adult rats.

This study examines whether treatment with GM1 ganglioside or the corticotropin (ACTH)(4-9) analogue ORG2766 can facilitate the behavioural recovery of adult rats with medial prefrontal cortex (mPFC) lesions, as animals are impaired in their food hoarding and spatial delayed alternation performance following mPFC lesions. No ameliorating effects of GM1 treatment on performance of these behaviours were observed. Although treatment with ORG2766 somewhat improved the hoarding performance of lesioned animals, the intermediate amount of pellets hoarded was not significantly different from that of either sham-operated or vehicle-treated lesioned rats. No effect of ORG2766 treatment was observed in the spatial delayed alternation test. Further, no changes were detected in the mesocortical dopamine innervation, presumed to be involved in the neural mechanism of behavioural sparing, in response to either treatment.

Monoamine concentrations in rat prefrontal cortex and other mesolimbocortical structures in response to partial neonatal lesions of the medial prefrontal cortex.

In an earlier study it was found that partial neonatal lesions of the medial prefrontal cortex (mPFC) resulted in an increased dopaminergic innervation in the remaining part of mPFC along with functional sparing. The present study assessed whether this response is restricted to this part of the cortex or whether also other structures of the mesolimbocortical system are involved. Furthermore, it was investigated whether the other monoaminergic systems were affected by neonatal mPFC lesions. In 6-day-old rats, the mPFC was partially ablated or a sham operation was made. The concentrations of dopamine (DA) and its metabolites increased to 250-350% in the remaining part of the mPFC compared to the sham-operated controls. The response was most prominent in this part of the cortex; no other mesolimbocortical structure showed such major changes of DA and its metabolites. In addition, a small increase in the concentrations of noradrenaline, serotonin and their metabolites was also spotted in the remaining mPFC and some other mesolimbocortical structures of the lesioned animals. The present data support the suggested involvement of DA in the neural mechanism of sparing of function, and this DA response seems to be most prominent in the remaining mPFC. However, the responses of the noradrenergic and serotonergic systems may also be important for sparing of function to occur.

Heterotopic Cortical Afferents to the Medial Prefrontal Cortex in the Rat. A Combined Retrograde and Anterograde Tracer Study.

Cortical afferent projections towards the medial prefrontal cortex (mPFC) were investigated with retrograde and anterograde tracer techniques. Heterotopical afferent projections to the medial prefrontal cortex arise in secondary, or higher order, sensory areas, motor areas and paralimbic cortices. On the basis of these projections three subfields can be discriminated within the mPFC. (1) The ventromedial part of mPFC, comprising the pre- and infralimbic areas, receives mainly projections from the perirhinal cortex. (2) The caudal two-thirds of the dorsomedial PFC, comprising frontal area 2 and the dorsal anterior cingulate area, receives projections from the secondary visual areas, the posterior agranular insular area and the retrosplenial areas. (3) The rostral one-third of the dorsomedial PFC is the main recipient of projections from the somatosensory and motor areas and the posterior agranular insular area. The laminar distribution of cells projecting to the mPFC varies considerably in the different cortical areas, just as the laminar distribution of termination of their fibres within the mPFC does. It is concluded that the corticocortical connections corroborate with subcortical connectivity in attributing to the mediodorsal projection cortex of the rat functions which are comparable to those of certain prefrontal, premotor and anterior cingulate areas in the monkey.

Activation of mesocortical dopaminergic system in the rat in response to neonatal medial prefrontal cortex lesions. Concurrence with functional sparing.

Neonatal lesions of the medial part of the rat prefrontal cortex (mPFC) (performed at the age of 6 days) resulted in a sparing in the performance of spatial delayed alternation (SDA) and an increase in dopaminergic (DA) innervation. The increased DA innervation was primarily observed in the remaining part of the mPFC. The DA fibre density was considerably higher in the non-ablated part of the mPFC, and the fibres were thicker with more large varicosities compared with sham-operated controls. Biochemical measurements showed a 3.5-fold increase in DA concentration in the remaining part of the mPFC of the animals with neonatal lesions when compared with the mPFC of sham-operated animals. In addition the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were increased. The metabolite/transmitter ratios, indicating DA utilisation, did not significantly differ from controls. The increased DA innervation and the increased concentration of DA and its metabolites in the animals with neonatal lesions further support our hypothesis that the mesocortical DA system is involved in the neural mechanism of sparing of function observed after neonatal mPFC lesions. However, sparing of function in animals with no discernable mPFC forces us to conclude that this DA response cannot be the only factor involved in the mechanism of sparing of function.

Spike-train analysis reveals "overshoot" in developing rat prefrontal cortex function.

A multivariate analysis of spontaneous single neuron firing in the developing prefrontal cortex (PFC) of urethane anesthetized rats has been made using selected spike-train parameters. In particular, the development of modal interspike intervals closely paralleled the volumetric 'overshoot' reported earlier by us for rat PFC. Thus, the growth phase is characterized by progressively higher firing rates associated with longer modal intervals (i.e., a change from phasic to tonic firing, suggestive of increasingly effective inhibition). In contrast, the subsequent abrupt reduction in PFC volume is accompanied by the appearance of extremely short interspike intervals with no concomitant change in overall mean discharge rates ('burst' firing). This last development could be largely due to the 'pruning' of excessive excitatory synaptic contacts.

Prognostic relevance of DNA flow cytometry in oligodendroglioma.

In a retrospective study of 85 cases, the prognostic value of DNA flow cytometry in oligodendrogliomas was evaluated. Paraffin-embedded material was processed for flow cytometry, and the survival rates of the patients with DNA diploid, aneuploid, and tetraploid tumors were compared using analysis of variance. In addition, the mitotic index was correlated with the results of flow cytometry. Finally, the results of flow cytometry, histopathologic grading, and counting mitoses were tested for dependency. Thirty-one percent of the tumors were diploid, 39% were tetraploid, and 31% were aneuploid. The results of the DNA flow cytometry did not correlate with the survival times (P = 0.798) or with tumor degree. In contrast, the number of mitoses (P less than 0.05), and the grades of the grading system of Smith (P less than 0.003) had relevance for the prognosis. No correlation between flow cytometry, histopathologic grading, and mitotic index was found. It is concluded that flow cytometry has no value in predicting the biologic behavior of oligodendrogliomas, whereas the number of mitoses is a valuable prognostic parameter and thus is considered to be incorporated into the grading system for oligodendrogliomas.

Prenatal development of neurons in the human prefrontal cortex. II. A quantitative Golgi study.

The quantitative development of neurons in the human dorsolateral and lateral prefrontal cortex was studied in Golgi-impregnated tissue from postmortem brains ranging from 13.5 weeks of gestation up to the second postnatal month. Pyramidal neurons in the future layers III and V of the cortical plate, as well as different types of neurons in the transient subplate zone, were studied. The basal dendrites of the future layer III and V pyramidal neurons show a slow increase during the first two-thirds of the period of gestation. From 27-32 weeks of gestation on, there is a rapid increase in the length of basal dendrites of layer III and V pyramidal neurons, while the number of basal dendrites per pyramidal neuron appears to stabilize at 26/27 weeks of gestation. The increase in total length of basal dendrites per pyramidal neuron is mainly due to an increase in the number of bifurcations and the growth of terminal segments. Throughout the whole period studied, the size of the layer III pyramidal basal dendritic tree was smaller than that of layer V pyramidal neurons. Thus, not until postnatal life do the layer III pyramidal basal dendrites become larger than those of layer V. No statistically significant differences were found for data of the pyramidal neurons between the superior and middle frontal gyri. The dendritic size of subplate neurons, except for the subplate inverted pyramidal neurons, significantly exceeds the size of the basal dendrites of the pyramidal neurons up to the seventh gestational month, which indicates an earlier maturation of these subplate neurons. During the period examined, no clear decrease in the size of the subplate neurons was observed. The present study shows that the dendritic parameters of either subplate or cortical plate pyramidal neurons rapidly increase during the periods of ingrowth of afferent fibers into the subplate zone and cortical plate, respectively. In the Golgi preparations of the prefrontal cortex, the size of the subplate neurons does not show any clearly regressive changes at the end of the prenatal period.

Neuroanatomical correlates of sparing of function after neonatal medial prefrontal cortex lesions in rats.

In rats, the possibility of neuroanatomical changes in response to partial medial prefrontal cortex lesions at postnatal day 6, concomitant with behavioural sparing, was investigated. The projections from the mediodorsal nucleus of the thalamus (MD) and the mesocortical dopaminergic (DA) projection were examined. No indications were found for a changed pattern of projection from MD in response to either a neonatal or an adult medial prefrontal cortex (mPFC) lesion. However, the DA innervation was changed after neonatal mPFC lesions. In the remainder of the mPFC, the DA fibre network proved to be denser, fibres were thicker, had more varicosities, and often the background staining was higher. None of these phenomena were seen in operated adult rats or in controls. It is postulated that the changes in DA innervation might contribute to the sparing of function observed in the spatial delayed alternation task.