Long‐term stability of axonal boutons in the mouse barrel cortex

Many lines of evidence indicate that postsynaptic dendritic spines are plastic during development and largely stable in adulthood. It remains unclear to what degree presynaptic axonal terminals undergo changes in the developing and mature cortex. In this study, we examined the formation and elimination of fluorescently‐labeled axonal boutons in the living mouse barrel cortex with transcranial two‐photon microscopy. We found that the turnover of axonal boutons was significantly higher in 3‐week‐old young mice than in adult mice (older than 3 months). There was a slight but significant net loss of axonal boutons in mice from 1 to 2 months of age. In both young and adult barrel cortex, axonal boutons existed for at least 1 week were less likely to be eliminated than those recently‐formed boutons. In adulthood, 80% of axonal boutons persisted over 12 months and enriched sensory experience caused a slight but not significant increase in the turnover of axonal boutons over 2–4 weeks. Thus, similar to postsynaptic dendritic spines, presynaptic axonal boutons show remarkable stability after development ends. This long‐term stability of synaptic connections is likely important for reliable sensory processing in the mature somatosensory cortex. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 252–261, 2016     

Ontogenesis of the spine-free initial zone of apical dendrites; studies in cortical pyramidal cells of albino rat]

1. In Golgi-Cox-impregnated coronal sections of albino rat brains at 1, 4, 26, 24, 30, 60 and 90 days it is presented the evolution of the spine-less, bare initial zone ("nude zone", NZ) at the proximal apical main dendrites of the layer V pyramidal neurons in the somatosensory and anterior limbie cortex. The quantitative results are analyzed by statistical methods. 2. The NZ is comprehended as a morphological correlate of the endodendritic neuroplasmic flow (Weiss 1944, Globus, Lux and Schuberl 1968, Kreutzberg 1973). The observed changes of the percental frequency and the average length of NZ increases rapidly. 3. The NZ occurs at first in the (12th) 16 postnatal day, thus in a time, when the organs of hearing and the eyes are differentiated completely. Between 16th and 24th day the percental frequency as well as the longitude of NZ increases. During this time the rats will be independent of the mother animals. With the full differentiation of the urogenital tract and especially with the sexual maturity the percentage frequency of NZ increases only at pyramidal cells in the anterior limbie cortex between 24th and 60th day. During 3rd month the NZ is occuring percental more frequently in the anterior limbic cortex than in the somatosensory cortex. At this time the average length of NZ is shorter in the limbic cortex. 4. As to the enriched, vivid movement of the animals and the playing impulse of the young rats the average length of NZ will be extended at pyramidal neurons in the somatosensory cortex during 2nd month, as well as the pattern of spine distribution will be changed along apical dendrites (Schlerhorn, unpublished). During the following (3rd) month the NZ will be shorteded in the somatosensory cortex, obviously caused by new formation of spines at the proximal main dendrites. 5. These newly formed spines in the initial zone of apical dendrites may be inhibitory spines. The inhibitory spines are stained only when using the mercury chromate impregnation according to Golgi-Cox, but not when using the silver chromate methods according to Golgi-Kopsch or Golgi-Bubenaite. The different tingibility of these spines by different Golgi techniques is discussed by Doedens, Nagel and Schierhorn (1974). The pyramidal neurons in the somatosensory cortex possess a longer average length of NZ (Lnz = 7,3[mum]) than the pyramidal cells in the anterior limbic cortex (Lnz = 6.2[mum]). As to NZ the differences between silver and mercury chromate stained pyramidal neurons are greater in the somatosensory cortex than in limbic cortex (see Tab. 7). Therefore we assume that there are in the initial zone of somatosensory pyramidal neurons more inhibitory spines than at the pyramidal neurons in the anterior limbic cortex. 6. The regional differences in the percentual frequency and in the average length of NZ between somatosensory and limbic cortex are new identifying marks of architectonic differentiation of the pyramidal neurons in cortical regions of phylogenetically different ages.     

Long-term effects of postnatal exposure to diethylstilbestrol on uterine estrogen receptor and growth.

Diethylstilbestrol (DES) treatment of female rats on postnatal days (PND) 1-5 reduces uterine growth, estrogen receptor (ER) level and gland number by PND 25, while daily DES treatment on PND 1-25 increases uterine growth 4-fold, further reduces ER level and completely suppresses gland formation. We now report the persistence of these effects in adults. By PND 60, uterine weight was 70% of controls in rats injected with DES on PND 1-5 but only 10% of controls in rats injected PND 1-10 or longer. In fact, uterine weights were the same on PND 10 and 60. Uterine gland numbers were reduced to 30% of controls in all DES-treated rats regardless of exposure length; however, luminal and glandular epithelial cell heights were reduced to less than 50 and 70%, respectively, of controls when DES was given on PND 1-25 but not when given on PND 1-5. Ovariectomy 7 days prior to sacrifice on PND 60 reduced uterine weight in controls by 67% and in rats injected with DES on PND 1-5 by 53%, but had no effect in rats injected with DES on PND 1-10. DES exposure at either PND 1-5 or 1-10 lowered ER levels by 35-50% at both 60 and 90 days. Treatment with a high dose of estradiol (E2) 1 week before sacrifice significantly down-regulated ER to the same concentration in all treatment groups at PND 60 and 90. Following E2 treatment, all groups also showed increased uterine weight at PND 60 and 90. These data show there is a short period of development (PND 5-10) in which further DES exposure indirectly inhibits uterine growth.     

Neonatal repetitive maternal separation causes long-lasting alterations in various neurotrophic factor expression in the cerebral cortex of rats

AimsThis study was carried out to examine the effects of early postnatal maternal separation stress on the development of the cerebral cortex with respect to time-dependent fluctuations of neurotrophic factor ligand and receptor expression.Main methodsWistar rats were separated from their mothers for 3 h per day during postnatal days (PND) 10 to 15. The cerebral cortex was analyzed by real-time RT-PCR for the evaluation of the expression of mRNA for brain-derived neurotrophic factor (BDNF), TrkB, insulin-like growth factor-1 (IGF-1), and type 1 IGF receptor (IGF-1R) on PND16, 20, 30, and 60.Key findingsThe expression of these neurotrophic factor ligands and receptors in the cerebral cortex was enhanced on PND16 and PND20, and then it returned to baseline levels on PND30. By PND60, however, the expression levels were attenuated.SignificanceThe important implication of this study is the persistent abnormal fluctuation of neurotrophic factor expression for a prolonged period, triggered even after the brain growth spurt. Given that neurotrophic factors play important roles in brain development, it can be speculated that the altered expression of these factors induced by maternal separation may interrupt normal brain development and ultimately lead to functional disruption. However, the possibility of such changes leading to various functional disruptions and the underlying mechanisms involved require further study.     

In vivo imaging detects a transient increase in brain arachidonic acid metabolism: a potential marker of neuroinflammation

In a rat model of neuroinflammation produced by an intracerebral ventricular infusion of bacterial lipopolysaccaride (LPS), we measured the coefficients of incorporation (k*) of arachidonic acid (AA, 20 : 4n-6) from plasma into each of 80 brain regions, using quantitative autoradiography and intravenously injected [1-(14)C]AA. Compared with control rats infused with artificial cerebrospinal fluid (aCSF), k* was increased significantly in 25 brain areas, many of them close to the CSF compartments, following 6-days of LPS infusion. The increases, ranging from 31 to 76%, occurred in frontal, motor, somatosensory, and olfactory cortex, thalamus, hypothalamus, and septal nuclei, and basal ganglia. Following 28 days of LPS infusion, k* was increased significantly in only two brain regions. Direct analyses of microwaved brain showed that 93 +/- 3 (SD) and 94 +/- 4% of brain radioactivity was in the organic extract as radiolabeled AA in the 6-day control and LPS-infused animals, respectively, compared with 91 +/- 3 and 87 +/- 6% in the 28-day control and LPS-infused animals. These results confirm that brain AA metabolism is disturbed after 6 days of LPS exposure, show this increase is transient, and that these changes can be detected and localized using in vivo imaging with radiolabeled AA.     

Lead-induced increase in antioxidant enzymes and lipid peroxidation products in developing rat brain

Pregnant rats were treated with 0.4% lead acetate through drinking water from 6th day of gestation and this treatment was continued till 21 post natal days (PND). Four regions of the brain namely hippocampus, cerebellum, frontal cortex and brain stem were dissected at 10, 20, 30 and 40 PND for estimation of lipid peroxidation products (LPP), catalase (CAT) and superoxide dismutase (SOD). The results indicate that there was a significant (P < 0.05) increase of LPP in exposed rats than their corresponding control at 10, 20 and 30 PND both in hippocampus and cerebellum. At PND 40, the LPP of control and exposed were found to be almost same in both the tissues indicating recovery from lead toxicity. CAT activity was significantly (P < 0.05) high in hippocampus of exposed rats up to PND 30 but up to PND 20 in cerebellum and frontal cortex. However, in brain stem, a significant (P < 0.05) increase in CAT activity was observed only at PND 10. A significant (P < 0.05) increase in SOD activity was observed up to PND 30 both in hippocampus and cerebellum on lead exposure. Frontal cortex exhibited a similar significant (P < 0.05) increase of SOD activity up to PND 20 and for brain stem up to PND 10. There was no significant change in the activity of antioxidant enzymes (CAT and SOD) and LPP in all the four brain tissues of control and exposed rats at PND 40 indicating recovery from lead-induced oxidative stress. This research work was presented as a poster in Annual Biomedical Research Conference for Minority Students (ABRCMS) at Dallas, Texas, USA, during November 10–13, 2004 and the abstract was printed on page 231 of the Conference Proceedings     

Effects of Age and Acute Ethanol on Glutamatergic Neurotransmission in the Medial Prefrontal Cortex of Freely Moving Rats Using Enzyme-Based Microelectrode Amperometry

Ethanol abuse during adolescence may significantly alter development of the prefrontal cortex which continues to undergo structural remodeling into adulthood. Glutamatergic neurotransmission plays an important role during these brain maturation processes and is modulated by ethanol. In this study, we investigated glutamate dynamics in the medial prefrontal cortex of freely moving rats, using enzyme-based microelectrode amperometry. We analyzed the effects of an intraperitoneal ethanol injection (1 g/kg) on cortical glutamate levels in adolescent and adult rats. Notably, basal glutamate levels decreased with age and these levels were found to be significantly different between postnatal day (PND) 28-38 vs PND 44-55 (p<0.05) and PND 28-38 vs adult animals (p<0.001). We also observed spontaneous glutamate release (transients) throughout the recordings. The frequency of transients (per hour) was significantly higher in adolescent rats (PND 28-38 and PND 44-55) compared to those of adults. In adolescent rats, post-ethanol injection, the frequency of glutamate transients decreased within the first hour (p<0.05), it recovered slowly and in the third hour there was a significant rebound increase of the frequency (p<0.05). Our data demonstrate age-dependent differences in extracellular glutamate levels in the medial prefrontal cortex and suggest that acute ethanol injections have both inhibitory and excitatory effects in adolescent rats. These effects of ethanol on the prefrontal cortex may disturb its maturation and possibly limiting individuals´ control over addictive behaviors.     

Development of long-term dendritic spine stability in diverse regions of cerebral cortex

Synapse formation and elimination occur throughout life, but the magnitude of such changes at distinct developmental stages remains unclear. Using transgenic mice overexpressing yellow fluorescent protein and transcranial two-photon microscopy, we repeatedly imaged dendritic spines on the apical dendrites of layer 5 pyramidal neurons. In young adolescent mice (1-month-old), 13%-20% of spines were eliminated and 5%-8% formed over 2 weeks in barrel, motor, and frontal cortices, indicating a cortical-wide spine loss during this developmental period. As animals mature, there is also a substantial loss of dendritic filopodia involved in spinogenesis. In adult mice (4-6 months old), 3%-5% of spines were eliminated and formed over 2 weeks in various cortical regions. Over 18 months, only 26% of spines were eliminated and 19% formed in adult barrel cortex. Thus, after a concurrent loss of spines and spine precursors in diverse regions of young adolescent cortex, spines become stable and a majority of them can last throughout life.     

Profile of acetylcholinesterase in brain areas of male and female rats of adult and old age

Inhibition of acetylcholinesterase (AChE)-metabolizing enzyme of acetylcholine, is presently the most important therapeutic target for development of cognitive enhancers. However, AChE activity in brain has not been properly evaluated on the basis of age and sex. In the present study, AChE activity was investigated in different brain areas in male and female Sprague-Dawley rats of adult (3 months) and old (18-22 months) age. AChE was assayed spectrophotometrically by modified Ellman's method. Specific activity (micromoles/min/mg of protein) of AChE was assayed in salt soluble (SS) and detergent soluble (DS) fractions of various brain areas, which consists of predominantly G1 and G4 molecular isoforms of AChE respectively. The old male rats showed a decrease (40-55%) in AChE activity in frontal cortex, striatum, hypothalamus and pons in DS fraction and there was no change in SS fraction in comparison to adult rats. In the old female rats the activity was decreased (25-40%) in frontal cortex, cerebral cortex, striatum, thalamus, cerebellum and medulla in DS fraction whereas in SS fraction the activity was decreased only in hypothalamus as compared to adult. On comparing with old male rats, old female rats showed increase in AChE activity in cerebral cortex, hippocampus and hypothalamus of DS fraction and decrease in hypothalamus of SS fraction. There was a significant increase in AChE activity in DS fraction of cerebral cortex, hippocampus, hypothalamus, thalamus and cerebellum in female as compared to male adult rats. However, no significant change in AChE activity was found in the SS fraction, except hypothalamus between these groups. Thus it appears that age alters AChE activity in different brain regions predominantly in DS fraction (G4 isoform) that may vary in male and female. These observations have significant relevance to age related cognitive deficits and its pharmacotherapy.     

Imaging the impact of cortical microcirculation on synaptic structure and sensory-evoked hemodynamic responses in vivo

In vivo two-photon microscopy was used to image in real time dendrites and their spines in a mouse photothrombotic stroke model that reduced somatosensory cortex blood flow in discrete regions of cortical functional maps. This approach allowed us to define relationships between blood flow, cortical structure, and function on scales not previously achieved with macroscopic imaging techniques. Acute ischemic damage to dendrites was triggered within 30 min when blood flow over >0.2 mm² of cortical surface was blocked. Rapid damage was not attributed to a subset of clotted or even leaking vessels (extravasation) alone. Assessment of stroke borders revealed a remarkably sharp transition between intact and damaged synaptic circuitry that occurred over tens of μm and was defined by a transition between flowing and blocked vessels. Although dendritic spines were normally ~13 μm from small flowing vessels, we show that intact dendritic structure can be maintained (in areas without flowing vessels) by blood flow from vessels that are on average 80 μm away. Functional imaging of intrinsic optical signals associated with activity-evoked hemodynamic responses in somatosensory cortex indicated that sensory-induced changes in signal were blocked in areas with damaged dendrites, but were present ~400 μm away from the border of dendritic damage. These results define the range of influence that blood flow can have on local cortical fine structure and function, as well as to demonstrate that peri-infarct tissues can be functional within the first few hours after stroke and well positioned to aid in poststroke recovery.