Selective Medial Septum Lesions in Healthy Rats Induce Longitudinal Changes in Microstructure of Limbic Regions, Behavioral Alterations, and Increased Susceptibility to Status Epilepticus.

Septo-hippocampal pathway, crucial for physiological functions and involved in epilepsy. Clinical monitoring during epileptogenesis is complicated. We aim to evaluate tissue changes after lesioning the medial septum (MS) of normal rats and assess how the depletion of specific neuronal populations alters the animals' behavior and susceptibility to establishing a pilocarpine-induced status epilepticus. Male Sprague-Dawley rats were injected into the MS with vehicle or saporins (to deplete GABAergic or cholinergic neurons; n = 16 per group). Thirty-two animals were used for diffusion tensor imaging (DTI); scanned before surgery and 14 and 49 days post-injection. Fractional anisotropy and apparent diffusion coefficient were evaluated in the fimbria, dorsal hippocampus, ventral hippocampus, dorso-medial thalamus, and amygdala. Between scans 2 and 3, animals were submitted to diverse behavioral tasks. Stainings were used to analyze tissue alterations. Twenty-four different animals received pilocarpine to evaluate the latency and severity of the status epilepticus 2 weeks after surgery. Additionally, eight different animals were only used to evaluate the neuronal damage inflicted on the MS 1 week after the molecular surgery. Progressive changes in DTI parameters in both white and gray matter structures of the four evaluated groups were observed. Behaviorally, the GAT1-saporin injection impacted spatial memory formation, while 192-IgG-saporin triggered anxiety-like behaviors. Histologically, the GABAergic toxin also induced aberrant mossy fiber sprouting, tissue damage, and neuronal death. Regarding the pilocarpine-induced status epilepticus, this agent provoked an increased mortality rate. Selective septo-hippocampal modulation impacts the integrity of limbic regions crucial for certain behavioral skills and could represent a precursor for epilepsy development.

Differentiation of white matter histopathology using b-tensor encoding and machine learning.

Diffusion-weighted magnetic resonance imaging (DW-MRI) is a non-invasive technique that is sensitive to microstructural geometry in neural tissue and is useful for the detection of neuropathology in research and clinical settings. Tensor-valued diffusion encoding schemes (b-tensor) have been developed to enrich the microstructural data that can be obtained through DW-MRI. These advanced methods have proven to be more specific to microstructural properties than conventional DW-MRI acquisitions. Additionally, machine learning methods are particularly useful for the study of multidimensional data sets. In this work, we have tested the reach of b-tensor encoding data analyses with machine learning in different histopathological scenarios. We achieved this in three steps: 1) We induced different levels of white matter damage in rodent optic nerves. 2) We obtained ex vivo DW-MRI data with b-tensor encoding schemes and calculated quantitative metrics using Q-space trajectory imaging. 3) We used a machine learning model to identify the main contributing features and built a voxel-wise probabilistic classification map of histological damage. Our results show that this model is sensitive to characteristics of microstructural damage. In conclusion, b-tensor encoded DW-MRI data analyzed with machine learning methods, have the potential to be further developed for the detection of histopathology and neurodegeneration.

Effects of perinatal undernutrition on social transmission of food preference in adult male Wistar rats.

Nutrition plays a fundamental role in learning and memory, and early experimental undernutrition interferes with brain memory processes. Social transmission of food preference (STFP) is a natural olfactory paired-associate learning test that has not been used to assess the effects of early undernutrition on memory consolidation. Male Wistar rats were randomly divided into two groups: control and early undernourished. The underfed rats received different percentages of a balanced diet during gestation. After birth, pups were underfed by alternating every 12 h between two lactating dams, one with ligated nipples. Weaning occurred on PD 25 followed by an ad lib diet until PD 90. Demonstrator rats were given powdered food mixed with cinnamon, followed by a 30-min interaction with an underfed observer. Thereafter, the observer had two choices of food: cinnamon or cocoa. During the food preference test, control and underfed OBS rats preferred the food containing cinnamon. Through social interaction, the UG OBS rats showed latency for head contacts and oral-nasal investigation was higher in the underfed rats; only head contacts and oral-nasal investigation frequency was lower; with the duration lower, but oral-nasal investigation duration was higher (p < 0.05). In the preference phase, the OBS underfed rat latencies for both stimuli were prolonged, the frequency lower only for cocoa, and the duration lower for cinnamon but higher for cocoa (p < 0.05). Findings suggested that early undernutrition interfered with the attentive social transmission to take a decision during the preference phase, but not with the short-term memory consolidation of social food preference.

Late-emerging effects of perinatal undernutrition in neuronal limbic structures underlying the maternal response in the rat.

Maternal care in the rat is an ancient behavioral response to specific multisensory inputs widely integrated in a complex forebrain, limbic and brain stem network to meet the basic needs of the young. Early undernutrition interferes with the morphofunctional organization of the brain, including maternal circuitry. The late-emerging effects of pre- and neonatal undernutrition on nest building and pup retrieval by lactating Wistar rats were correlated with dendritic arbor and perikaryon measurements (Golgi-Cox) in layer II pyramidal neurons of the anterior cingulate cortex, layer III pyramidal neurons of the medial prefrontal cortex and multipolar basolateral amygdala neurons examined on lactation days 4 and 12. In the underfed group, pregnant F0 dams received different percentages of a balanced diet. After birth, prenatally underfed (F1) pups continued the undernutrition by remaining with a nipple-ligated mother for 12 h. Weaning occurred at 25 days of age, and pups were subsequently provided an ad libitum diet. At 90 days of age, F1 dams were maternally tested. Early underfed dams showed significant reductions in nest building and prolonged retrieval latencies for grasping pups by inappropriate body areas. The behavioral alterations were concurrent with highly significant reductions in the somatic cross-sectional area and perimeter, spine density and dendritic crossings of cingulate cells and medial prefrontal cortical pyramids, as well as smaller effects on amygdala neurons. The anatomical findings suggest different postsynaptic organizations that may affect the neuronal excitability stages for the integration and encoding of cues triggering the altered maternal response components of early underfed dams.

Effects of pre- and neonatal undernutrition on the kyphotic response and c-Fos activity in the caudal periaqueductal gray of primiparous lactating Wistar rats.

In rodents, the most representative component of maternal behavior that meets the purpose of newborn nutrition is the kyphotic posture. During this posture, the mother maintains a unique environment for the protection, thermal regulation and breast-feeding of the progeny. The aim of this study was to investigate possible deficiencies in the kyphotic posture of adult lactating dams with pre- and neonatal undernutrition evoked by their own pups suckling in a home-cage situation. Wistar dams that had been previously exposed to perinatal undernutrition were mated at 90days of age, and pregnancy was confirmed by vaginal smears. Before testing if the perinatal underfed dam affected behavior, pups were removed (4h), and both the maternal response and the kyphotic posture were video-recorded (1h) and analyzed at 4 and 12days of lactation. Pre- and post-test litter weight gain was obtained. To immunostain the caudal periaqueductal gray, the litter was separated from their dams 24h before suckling stimulation. The results showed that underfed dams significantly reduced the duration of high kyphosis by choosing unconventional postures (prone and partial kyphosis). The body weight of the F1 offspring was significantly reduced, and the underfed F0 dams showed reduced c-Fos immunostaining at the caudal periaqueductal gray. The findings showed that early underfed dams have deficiencies in the mechanisms underlying the kyphosis, possibly because the pups' cues to evoke this posture were suboptimal and/or because the dam expressed deficient nursing. The results suggest that the abnormal kyphotic posture may affect the mother-litter bonds and have long-term effects on neonatal brain functions.

Maternal alterations induced by exposure to an unfamiliar home cage in early underfed dams.

The expression of different behavioral components in the adult rat depends on a number of early influences, including age, hormones, manipulations of sensory cues, and perinatal undernutrition, all of which impact the development of brain areas underlying adaptive processes, maternal behavior, and the response to novelty. The current study investigates the effects of pre- and neonatal undernutrition on various components of maternal behavior of dams exposed to the challenge of an unfamiliar home cage on days 4, 8, and 12 of lactation. Food restriction was initiated from gestational day (G) G6 to G19 when dams received 50-70% of the normal balanced diet, followed by 100% from G20 to G21. After birth, pups were underfed by alternating every 12h between two lactating dams, one of which, had ligated nipples. Weaning was at 25days of age followed by an ad libitum diet until postpartum day 90, when females were mated, and subsequently tested for maternal behavior in an unfamiliar cage. The results indicated that in early underfed mothers the frequency of handling wood shavings and of, approaching, licking, crouching, and grasping pups for retrieval was significantly reduced. Moreover, self-grooming increased substantially in the underfed dams, but the frequency of rearing was reduced. Additionally, the body weight of pups nursed by early underfed dams was significantly lower than that of control pups. These findings suggest a relation between early food restriction and the deficient maternal care observed when these dams were challenged by exposure to an unfamiliar home cage.

Effects of perinatal undernutrition on the circumvallate papilla of developing Wistar rats.

During the gestation and the lactating periods the gustatory papillae contain taste buds that respond to different flavors and aversive stimuli. The current study analyzed the effects of pre-and neonatal undernutrition on the circumvallate papillae of rats at 12, 20, and 30days of age. Early undernourishment occurred from gestational days G6 to G19 when dams received low percentages of food followed by a balanced diet from G20-21. After birth pups were underfed by rotating two lactating dams every 12h; in one of them, the nipples were tied. The pups were weaned at 25days of age, and then given an ad libitum diet. Under anesthesia the tongues were removed and stained with the hematoxylin-eosin (H-E) procedure. The results indicated that young underfed rats had significantly body weight reductions. The tongue measurements in underfed rats showed reduced total area and length of the anterior portion, but there were negligible effects on the posterior portion. The circumvallate papillae in underfed rats was significantly reduced in major length, major diameter, and total and upper areas, but unaffected in the lateral wall trench region. The taste bud areas and minor diameter were unaffected by undernutrition, but there were significant reductions in the total number of visible taste buds and the major diameter, delayed opening of taste bud pores, and an increased number of closed pores were also observed. These alterations by undernutrition reflect the vulnerability of structures in the gustatory oral cavity and suggest a possible interference with the receptors activation, and transduction and perhaps with the taste encoding of signals to generate the gustatory sensory and hedonic responses.

Effects of perinatal undernutrition on the basilar dendritic arbor of the anterior cingulate pyramidal neurons in lactating dams.

In altricial species, early pre- and neonatal undernutrition interferes with the neuronal organization of several brain structures that have critical time windows for synaptic organization, including the prefrontal cortex. In Golgi-Cox stained tissue the basilar dendritic arbor of pyramidal neurons in the anterior cingulate cortex of early underfed adult lactating dams was evaluated. The anterior cingulate of the rat plays a major role in the execution of sexual, maternal and visual attentional control and other cognitive responses. The effects of neonatal undernutrition on the basilar dendritic tree and perikaryon measurements in layer II/III pyramidal neurons of the anterior cingulate were examined in lactating dams at postpartum days 8 and 12. In the underfed dams the distal portions of the basilar dendrites had fewer branches and a lower dendritic density of dendrites, and neurons had perikarya with reduced perimeter and cross-sectional area. Thus, the neuronal alterations may interfere the plastic synaptic activity and with maternal cognitive performance of rats subjected to early underfeeding. These anatomical alterations of the anterior cingulate may help to understand the disruption of long-term cognitive processes associated with perinatal food restriction.

Effects of perinatal undernutrition on the development of neurons in the rat insular cortex.

The insular cortex (IC) of the rat is a major area for the convergence and integration of olfactory, gustatory, and visual information, and at present it is unclear if perinatal undernutrition interferes with the structure and function of the IC neurons. Golgi-Cox-stained cells of the IC were studied in control and undernourished Wistar rats at 12, 20, and 30 days of age. Pregnant dams were undernourished by the reduction of a balanced diet during a part of the gestational period (G6-G18). After parturition (P1-P23) pups remained for 12 hours with a normal and 12 hours with a nipple-ligated dam. Undernutrition significantly reduced the number, and the arborization of the dendritic arbors, and the perimeter, and cross-sectional area of perikarya. The IC neuronal morphology appearances suggest a possible mechanism for the impairment in information processing of complex phenomena such as taste sensation and hedonic response.

Development of mitral cells and olfactory bulb layers in neonatally undernourished rats.

Cell alterations in the central nervous system are consistent consequences of early undernourishment. Because little is known about the effects of neonatal udernourishment upon the main olfactory bulb (OB) in Golgi-Cox stained material, we evaluated the total OB cross-sectional area, the area of individual OB layers, and the area of type II mitral cells perikarya and their dendritic processes in undernourished Wistar rats of 7, 14, and 21 days of age. Data showed that neonatal undernourishment reduced both the OB and the individual layers areas; minimal perikarya effects and significant reductions in the number and extension of MC dendrites. Although macroneurons are formed prenatally, neonatal undernourishment at critical periods may have long-lasting effects that interfere with the functional maturity of the OB. These findings may have relevant consequences for early odor discrimination of the offspring, since olfaction is a fundamental sensorial avenue for newborn adaptive responses and maternal care.

Movement activity recovers the loss of spines owing to chronic immobilization.

In chronically movement-restricted Wistar rats, we described a significant decrease of spines along apical shafts of layer V cortical pyramids. Current study indicates that the liberation at 40 days of rats whose movements had been restricted since 20 days of age produces a gradual recovery of the number of spines, reaching the control values at 80 days of age and that this process occurred faster in the motor than in the sensory cortices. Nevertheless, when R(20) rats were liberated at 80 days, the number of spines had not fully recovered when rats were 120 days old. Spine recovery is a form of cortical experience-dependent plasticity.

Contributions of undernutrition and handling to huddling development of rats.

When newborn rats are separated from the mother, they consistently exhibit the huddling response to maintain body temperature and physical contact. Therefore, we investigated if preweaning handling/sensory stimulation may overcome the huddling deficiencies associated to neonatal undernourishment/maternal deprivation of Wistar rats maintained at constant temperature (30 degrees C). The data indicated that initial and final temperatures in the pile of undernourished (U) and undernourished stimulated (Us) pups was reduced compared to their controls (C and Cs, respectively). Huddling latency was prolonged at 5 days of age in the Us group and at 20 days of age in the U pups. On postpartum day 5, U and Us subjects were similar in battery and pile-huddling performance compared to their controls; thereafter, the frequency of battery type was low and pile type was high (in frequency) in all experimental treatments. The frequency of recycling from the pile in the Us pups in most of the ages was significantly reduced compared to U and C subjects, suggesting that early sensory stimulation possibly accelerates the maturation of thermoregulatory brain structures underlying the huddling response and causing increased physical contacts. The data provide evidence that both neonatal undernutrition/maternal deprivation and early sensory stimulation may modify the huddling response by reducing or increasing, respectively, brain mechanisms underlying huddling. The amount of physical contact the newborns receive from their littermates and the mother may be a fundamental source of sensory cues for neuronal maturation and brain functioning.

Organization of olfactory glomeruli in neonatally undernourished rats.

Newborn rats maintain mother-litter bonds by using olfactory signals. At birth, units in the olfactory glomeruli (OG) are immature and vulnerable to noxious epigenetic factors like undernutrition. Because little is known about the effects of neonatal undernutrition upon the OG morphological organization, different OG parameters were studied in undernourished Wistar rats at 7, 14 and 21 days of age. The issue was addressed by analyzing the olfactory bulb (OB) cross sectional area, the total number and area of OGs in the OB coronal sections, and the distribution of OG area in dorsal and ventral quadrants. Reductions in the OB and OG cross sectional areas were detected at 7 and 14 days posnatally. OG area comparisons by OB quadrants were reduced along the study in quadrants, with larger effects in medial than in lateral OB quadrants. Current OG cytoarchitectonic modifications may affect the newborn capabilities for odour discrimination by disrupting early mother-litter interactions.

Neonatal food restriction and binaural ear occlusion interfere with the maturation of cortical motor pyramids in the rat.

Golgi-Cox-impregnated pyramidal neurons of layer five motor cortical area were investigated in control, binaural ear-occluded control, undernourished and binaural ear-occluded undernourished Wistar rats of 12, 20 and 30 days of age. In neonatally undernourished, binaural ear-occluded-undernourished and partly in ear-occluded-control subjects, there were significant reductions in both the number and extent of the distal part of the dendritic branches of motor pyramids compared to their controls. Moreover, minimal effects on perikarya measurements were observed. These findings suggest that neonatal undernutrition and the concurrent reduction of auditory cues affect dendritic arbor development and possibly the convergence of the auditory experience upon motor pyramids and may interfere with the neocortical modulation of postural and movements activities.

Alterations in the solitary tract nucleus of the rat following perinatal food restriction and subsequent nutritional rehabilitation.

Newborn of altricial species maintain functional gustatory communication with the mother because the neural substrate and the capacity to discriminate and promote gustofacial responses are already operating. Because little is known about the effects of perinatal food restriction upon gustatory neuronal brain stem structures, we characterized neuronal Golgi-Cox alterations of the solitary tract rostral portion (NSTr) where gustatory information is known to convey in neonatal Wistar rats. Pre-and neonatally undernourished rats exhibited a general reduction in the number and extension of distal dendrites particularly in small neurons but little effect upon perikarya measurements of the NSTr neuronal population. By contrast, in nutritional and sensory rehabilitated rats the number of distal dendrites increased, although the dendritic extensions were less affected compared to perinatally underfed and control subjects. The data indicate that perinatal food restriction interferes with the NSTr dendritic arbor organization, while nutritional and sensorial rehabilitation given by normally lactating dams induced plastic changes presumably modifying the integrative processes underlying early taste discriminative capabilities. Moreover, since perinatal food restriction is a powerful stressor influence and the NST forms a part of a complex system underlying adaptive stress responses, the neuronal alterations observed here may be partly due to this noxious perinatal influence.

Retrieving of pups by neonatally stressed mothers.

In the rat, perinatal food and maternal deprivation provoke long-lasting effects upon the retrieving responses of dams to displaced pups. In the current study, the retrieving latency and the disruption in the body area of pups chosen by the mother to transport them to a new location was investigated on days 4, 8 and 12 postpartum in lactating Wistar rats. Rats, neonatally underfed by daily (12 h) mother-litter separation in an incubator from days 1 to 23 postpartum, exhibited prolonged retrieving latencies and disruption in the body area of young ones chosen by the dam to transport them to the nest. Furthermore, neonatally underfed dams frequently transported pups in a rude manner eliciting sonic distress cries from them compared to control mothers. These findings are possibly relevant to understand the impact of epigenetic influences on offspring brain and physiological maturation partly mediated through maternal care.

The hindlimb hyperextension associated to the Urogenital response in newborn undefed rats

Background. The aim of the study was to investigate how neonatal undernutrition interacts with mother-infant relationship to interfere with the expression of the urogenital response in the newborn. Methods. The hyperextensive reflex components associated to the urogenital response (HUR) were measured between postnatal days 1-21, in control and neonatally undernourished rats with or without fullness of the bladder. Results. In Experiment 1, both male and female underfed rats with different degress of bladder fullness exhibited an increment in HUR latency and vertical hindlimb displacement, reduced transversal separation and prolonged performance of hindlimb relaxation. Experiment 2 was performed in rats after urine elicitation provoked only reduced hindlimb transversal separation in males, and prolonged latency to HUR in females. Discussion. These findings may be related to the vulneability to neonatal undernutrition of the maturational processes which take place in the spinal cord, the condition of the bladder, and hinleg muscle maturation during this period of life. Conclusions. Data suggest that neonatal undernutrition may play a role in mother-infant interaction by interfering with HUR responses to maternal anogenital licking of pups

Alterations in the thalamic reticular nucleus of rats neonatally treated with thyroxine

En ratas Wistar normales y tratadas neonatalmente con tiroxina, se obtuvieron cortes seriados del tálamo, teñidos con el método de Golgi-Cox en las edades de 12,20 y 30 días. En la zona de núcleo reticular talámico (TRN), se contó el número de neuronas visibles, el área y la máxima extensión transversal del TRN en un total de 120 secciones. Los hallazgos indicaron que con relación al numero de neuronas en los animales tratados con T4, ocurrió un incremento signifiativo inicial de ellas a los 12 días de edad, seguido de un decremento igualmente significativo a los 20 y 30 días postnatales. Con respecto al área y a la máxima extensión transversal del TRN, sólo se observó una reducción progresiva que alcanzó sus valores más bajos a los 30 días de edad, sin ocurrir el incremento inicial que se ha descrito para el tejido neuronal. Los hallazgos sugieren que el tratamiento neonatal con T4, pudiera interferir con el desarrollo neuronal del TRN y, asimismo, a largo plazo, con las funciones modulatorias sensoriales del TRN