Dendritic morphology in the striatum and hypothalamus differentially exhibits experience-dependent changes in response to maternal care and early social isolation

Changes in neuron morphology, stemming from experiences in early life or adulthood, may be the basis for changes in behavior and their underlying functional mechanisms. For example, reproductive experience has been shown to significantly alter neuron morphology in the hippocampus and prefrontal cortex. In contrast to the effects of reproductive experience, a form of enrichment, on neuron morphology, our understanding of the effects of early social isolation on adult neuron morphology is limited. Therefore, the present study examined changes in neuron morphology in the dorsal (caudate nucleus) and ventral (nucleus accumbens, shell region) striatum and the medial preoptic area of adult virgin and postpartum females exposed to either artificial or maternal rearing during development. Primary results show that regardless of early social isolation, neurons in the caudate nucleus of postpartum females have decreased dendritic complexity compared to virgin females. Maternal experience also increased dendritic complexity in neurons of the nucleus accumbens shell. However, both early social isolation and maternal experience in adulthood influenced dendritic complexity in the medial preoptic area. Together these findings suggest that hypothalamic and striatal neurons show experience-dependent dendritic plasticity and the type and timing of these experiences differentially affect the location and degree of these morphological changes.     

Oestrogen-independent, experience-induced maternal behaviour in female mice

Nulliparous female mice that have not experienced mating, pregnancy or parturition show near immediate spontaneous maternal behaviour when presented with foster pups. The fact that virgin mice display spontaneous maternal behaviour indicates that the hormonal events of pregnancy and parturition are not necessary to produce a rapid onset of maternal behaviour in mice. However, it is not known how similar maternal behaviour is between virgin and lactating mice. In the present study, we show that naturally postpartum females are faster to retrieve pups and spend more time crouching over pups than spontaneously maternal virgin females, and that these differences diminish with increased maternal experience. Moreover, 4 days of experience with pups induced pup retrieval on a novel T-maze. Furthermore, the effects of experience on subsequent maternal responsiveness are not dependent on gonadal hormones because ovariectomised females with 4 days of pup experience show pup retrieval on a novel T-maze similar to that of postpartum mice. Four days of maternal experience also induced T-maze pup retrieval in ovariectomised aromatase knockout female mice that was not significantly different from the maternal responsiveness of ovariectomised wild-type littermates. These data suggest that maternal experience can induce maternal behaviour in females that have never been exposed to oestradiol at any time in development or adulthood. Finally, ovariectomised pup-experienced females continue to retrieve pups on a novel T-maze 1 month after the initial experience, suggesting that, even in the absence of oestradiol, maternal experience produces long-lasting modifications in maternal responsiveness.     

Functional mapping of the neural circuitry of rat maternal motivation: effects of site-specific transient neural inactivation

The present review focuses on recent studies from our laboratory examining the neural circuitry subserving rat maternal motivation across postpartum. We employed a site-specific neural inactivation method by infusion of bupivacaine to map the maternal motivation circuitry using two complementary behavioural approaches: unconditioned maternal responsiveness and choice of pup- over cocaine-conditioned incentives in a concurrent pup/cocaine choice conditioned place preference task. Our findings revealed that, during the early postpartum period, distinct brain structures, including the medial preoptic area, ventral tegmental area and medial prefrontal cortex infralimbic and anterior cingulate subregions, contribute a pup-specific bias to the motivational circuitry. As the postpartum period progresses and the pups grow older, it is further revealed that maternal responsiveness becomes progressively less dependent on the medial preoptic area and medial prefrontal cortex infralimbic activity, and more distributed in the maternal circuitry, such that additional network components, including the medial prefrontal cortex prelimbic subregion, are recruited with maternal experience, and contribute to the expression of late postpartum maternal behaviour. Collectively, our findings provide strong evidence that the remarkable ability of postpartum females to successfully care for their developing infants is subserved by a distributed neural network that carries out efficient and dynamic processing of complex, constantly changing incoming environmental and pup-related stimuli, ultimately allowing the progression of appropriate expression and waning of maternal responsiveness across the postpartum period.     

Autoradiographic analysis of GABAA receptor binding in the neural anxiety network of postpartum and non-postpartum laboratory rats

Postpartum female rats exhibit a suppression of anxiety-related behaviors when compared to diestrous virgin females, pregnant females, and males. This blunted anxiety promotes optimal maternal care and involves elevated GABA neurotransmission, possibly including greater density of GABA(A) and benzodiazepine receptors in the postpartum brain. We here examined autoradiographic binding of [(3)H]muscimol to measure the total population of GABA(A) receptors and [(3)H]flunitrazepam to assess density of benzodiazepine sites in the medial prefrontal cortex, bed nucleus of the stria terminalis, amygdala, hippocampus, and periaqueductal gray of female rats sacrificed on day 7 postpartum, day 10 of pregnancy, or as diestrous virgins. A group of sexually na?ve male rats was also included. We found that [(3)H]muscimol binding did not differ among groups in any site but that diestrous virgin females had greater [(3)H]flunitrazepam binding in the CA1 and dentate gyrus of the hippocampus compared to mid-pregnant females and males. Notably, postpartum and diestrous virgin females did not significantly differ in binding of either ligand in any site examined. This is the first study to evaluate the densities of GABA(A) and benzodiazepine binding sites simultaneously across three female reproductive states and sex with a focus on brain sites influencing anxiety-related behaviors. The results suggest that changes in other GABA(A) receptor characteristics such as subunit composition, or increased presynaptic GABA release during interactions with offspring, must instead play a greater role in the postpartum suppression of anxiety in laboratory rats.     

Morphological heterogeneity within the cingulate cortex in rat: a horseradish peroxidase transport study.

We compared the connections of two areas within rat cingulate cortex, the Cg1/Cg2 area vs the Cg3 area, by iontophoresing small quantities of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) into either of these two divisions and identifying afferent and efferent connections. Cortical projections were more widespread for the cingulate cortex (Cg3) area than for the Cg1/Cg2 area and included the dysgranular and agranular insular cortex, and perirhinal cortex. The Cg3 area received input from the CA1 layer of the hippocampus while the Cg1/Cg2 area was interconnected primarily with retrosplenial cortex. In the brainstem, both received input from Barrington's nucleus however, many of the subcortical connections of the two areas differed and supported the hypothesis that the Cg3 area is part of the limbic and visceral motor system while the Cg1/Cg2 area is more closely allied with somatic motor control. The Cg3 area received input from the basolateral nucleus of the amygdala, the supramammillary hypothalamic nucleus, the laterodorsal tegmental nucleus, and the lateral parabrachial nucleus. The Cg1/Cg2 area received input from the substantia nigra and targeted deep layers of the superior colliculus. Thus, rat cingulate cortex is a heterogeneous area that can be further subdivided into separate limbic/autonomic (Cg3) and somatic motor areas (Cg1/Cg2).     

Does prenatal nicotine exposure alter the brain's response to nicotine in adolescence? A neuroanatomical analysis

This study examined changes in dendritic morphology and spine density in multiple brain regions [Zilles' areas: (i) the Cg3 region of the anterior cingulate cortex or the medial prefrontal cortex, layer III (Cg3); (ii) the dorsal agranular insular cortex, layer III (AID); (iii) the PAR I region of the parietal cortex, layer III (Par1) and (iv) the nucleus accumbens (NAc)]of Long–Evans rats following exposure to nicotine prenatally, in late adolescence, or both prenatally and in adolescence. Prenatal nicotine exposure induced enduring changes in neuroanatomical organisation that varied between male and female offspring, with males exhibiting increased dendritic complexity of neurons in AID and NAc whereas females experienced increased dendritic complexity in Par1 but decreased dendritic complexity of neurons in NAc. Similarly, nicotine given in late adolescence dramatically reorganised neural circuitry of both male and female offspring, with males exhibiting decreased dendritic complexity of neurons in Par1 and Cg3 but increased dendritic complexity in AID, and females exhibiting decreased dendritic complexity in Cg3 and NAc but increased complexity in AID. Exposure to nicotine both prenatally and in adolescence produced few neuroanatomical parameters that demonstrated a prenatal experience × adolescent drug administration interaction. Females showed additive effects in Par1, Cg3 and NAc whereas males demonstrated additive effects only in AID. Thus, the timing of nicotine exposure produced differential effects on cerebral organisation in a regionally specific manner.     

Cortical expression of glial fibrillary acidic protein and glutamine synthetase is decreased in schizophrenia

Altered expression of structural and functional molecules expressed by astrocytes may play a role in the pathophysiology of schizophrenia. We investigated the hypothesis that the astrocytic enzyme glutamine synthetase, involved in maintaining the glutamate-glutamine cycle, and the cytoskeletal molecule glial fibrillary acidic protein (GFAP) are abnormally expressed in schizophrenia. We used Western blot analysis to measure levels of glutamine synthetase and GFAP in several brain regions of subjects with schizophrenia and a comparison group. We found that glutamine synthetase protein expression was significantly decreased in the superior temporal gyrus, and both glutamine synthetase and GFAP were significantly reduced in the anterior cingulate cortex in schizophrenia. Neither molecule demonstrated altered expression in the dorsolateral prefrontal cortex, primary visual cortex, or hippocampus. Chronic treatment with haloperidol did not alter the expression of these molecules in the rat brain, suggesting that our findings are not due to a medication effect. These data support an astrocytic component to the pathophysiology of schizophrenia and suggest that astrocytic molecules involved in enzymatic activity and cytoskeletal integrity may have a role in disease-related abnormalities in this illness.     

Maternal behavior: activation of the central oxytocin receptor system in parturient rats?

Parturition plays a critical role in the full expression of maternal behavior in postpartum females, yet the precise mechanism remains unclear. Here we examined the role of parturition in the activation of Fos and FosB in the central oxytocin receptor (OTR) system in rats. Although expression of FosB, not Fos, was seen in the piriform cortex (Pir) and caudate putamen of virgin and pregnant females, activation of Fos and FosB with extensive co-localization was found in the medial preoptic area, the bed nucleus of the stria terminalis and Pir of parturient brain. This parturition induced activation of Fos and FosB was identified in the central OTR-expressing cells as well as in oxytocinergic neurons. Our data provide direct evidence, for the first time, that parturition activates Fos and FosB in the central OTR system. We propose that Fos and FosB may have comparable functions on initiating maternal behavior at parturition.     

Blockade of Basic Fibroblast Growth Factor Retards Recovery from Motor Cortex Injury in Rats

The endogenous expression of basic fibroblast growth factor (bFGF) was blocked by neutralizing antibodies following unilateral suction lesions of the motor cortex. Rats with control treatment (saline, goat IgG) after motor cortex lesions showed slow recovery of forelimb manipulatory abilities. Rats with blockade of bFGF expression showed little recovery. Anatomically, the control-treated lesioned rats showed an acute increase in bFGF and glial fibrillary acidic protein (GFAP) reactivity, and chronically they had normal dendritic arborization and spine density in layer V pyramidal cells in the remaining motor cortex. In contrast, rats treated with antibodies to bFGF showed little bFGF reactivity, normal GFAP reactivity, and atrophy of dendritic arbor and decreased spine density in layer V pyramidal cells. These results demonstrate the importance of endogenous bFGF release in processes related to functional recovery after cortical injury.     

Pain-related aversion induces astrocytic reaction and proinflammatory cytokine expression in the anterior cingulate cortex in rats

Pain involves sensory and affective dimensions. It is well-known that activation of glial cells and a subsequent increase in proinflammatory cytokines contribute to the pathogenesis of pain sensation. However, the role of glial cells and proinflammatory cytokines in pain affect is unclear. Several lines of evidence indicate that the anterior cingulate cortex (ACC) is a key structure for pain affect. Using the formalin-induced conditioned place avoidance (F-CPA) model, which reflects the pain-related negative affective state induced by nociceptive stimuli, we examined the mRNA and protein expression levels of astrocytic markers and proinflammatory cytokines in the ACC. F-CPA produced robust aversion-like behaviors in rats. In parallel, a significant increase of mRNA of astrocytic markers (GFAP and S100B), and proinflammatory cytokines (IL-1β and TNF-α) were observed in the ACC. The protein level of GFAP, IL-1β and TNF-α were also enhanced in the ACC. The results showed for the first time that astrocytes and proinflammatory cytokines are associated with the processing of pain-related aversion and may be crucial players in the affective dimension of pain in rats.     

Topographic organization of efferent projections of medial frontal cortex

By using fluorescent retrograde tracers, we compared efferent projections of the medial frontal cortex to two subcortical areas: the superior colliculus, a somatic motor area, and the laterodorsal tegmental nucleus, a visceral motor area. Neurons projecting to the superior colliculus originated in layer V of the cingulate (Cg1 area) and medial agranular cortex, while neurons projecting to the laterodorsal tegmental nucleus originated in layers V and VI of the cingulate (Cg3 area) and infralimbic cortex. Thus, within the medial frontal cortex, the ventral portion (the Cg3 and infralimbic areas) may be a visceral motor area while the dorsal portion is a somatic motor region.     

Skilled reaching training promotes astroglial changes and facilitated sensorimotor recovery after collagenase-induced intracerebral hemorrhage

Spontaneous intracerebral hemorrhage (ICH) is the most devastating type of stroke and a leading cause of disability and mortality worldwide. Although rehabilitation improves recovery after ICH the cellular mechanisms involved are poorly understood. We decided to examine if skilled (SK) and unskilled (US) training after sham or intracerebral hemorrhage (ICH) surgeries would induce GFAP+ astrocytic changes and whether these modifications can be associated with functional improvement. A 4-week course of motor training, involving either skilled and unskilled training began seven days after surgery; sensorimotor recovery was evaluated using Staircase, ladder walk and cylinder tests. Histological and morphometric analyses were used to assess GFAP+ cell bilaterally in forelimb sensorimotor cortex and dorsolateral striatum. All behavioral tests showed that ICH-SK rats experienced a greater degree of recovery when compared to ICH no task or ICH-US groups; no behavioral differences were found among all sham groups. Astrocytic density was increased in all analyzed structures for ICH no task, ICH-SK and ICH-US rats. Morphological analysis revealed an increased number of primary processes in ipsilateral (to lesion) sensorimotor cortex for all ICH groups. Present results also revealed that both ICH and SK induced an increased length of GFAP+ primary process; there was a further increase in length processes for ICH-SK group in sensorimotor cortex and ipsilateral striatum. We suggest that skilled reaching is an effective intervention to promote astrocytic plasticity and recovery after ICH.     

Astrocyte‐specific insulin‐like growth factor‐1 gene transfer in aging female rats improves stroke outcomes

Middle aged female rats sustain larger stroke infarction and disability than younger female rats. This older group also shows age‐related reduction of insulin like growth factor (IGF)?1 in serum and in astrocytes, a cell type necessary for poststroke recovery. To determine the impact of astrocytic IGF‐1 for ischemic stroke, these studies tested the hypothesis that gene transfer of IGF‐1 to astrocytes will improve stroke outcomes in middle aged female rats. Middle aged (10‐12 month old), acyclic female rats were injected with recombinant adeno‐associated virus serotype 5 (AAV5) packaged with the coding sequence of the human (h)IGF‐1 gene downstream of an astrocyte‐specific promoter glial fibrillary acidic protein (GFAP) (AAV5‐GFP‐hIGF‐1) into the striatum and cortex. The AAV5‐control consisted of an identical shuttle vector construct without the hIGF‐1 gene (AAV5‐GFAP‐control). Six to eight weeks later, animals underwent transient (90 min) middle cerebral artery occlusion via intraluminal suture. While infarct volume was not altered, AAV5‐GFAP‐hIGF‐1 treatment significantly improved blood pressure and neurological score in the early acute phase of stroke (2 days) and sensory‐motor performance at both the early and late (5 days) acute phase of stroke. AAV5‐GFAP‐hIGF‐1 treatment also reduced circulating serum levels of GFAP, a biomarker for blood brain barrier permeability. Flow cytometry analysis of immune cells in the brain at 24 hr poststroke showed that AAV5‐GFAP‐hIGF‐1 altered the type of immune cells trafficked to the ischemic hemisphere, promoting an anti‐inflammatory profile. Collectively, these studies show that targeted enhancement of IGF‐1 in astrocytes of middle‐aged females improves stroke‐induced behavioral impairment and neuroinflammation.     

Affect and nurturance: mechanisms mediating maternal behavior in two female mammals

This review will discuss hormonal and psychological factors involved in the initiation and maintenance of maternal behavior during the postpartum period in rat and human mothers. Research on primiparous rats suggests that among the ways hormones act to promote maternal responsiveness are by increasing the mother's attraction to odors of young pups, decreasing her natural neophobia and fearfulness, and increasing the ease with which experiences obtained during initial mother-young contacts are retained. Long-term maintenance of elevated maternal responsiveness in the rat is not directly under hormonal control but instead depends on a minimal period of direct interaction with young during the early postpartum. In human first-time mothers, the onset of maternal responsiveness is also directly affected by their mood state as well as their attraction to infants; these factors are, in turn, influenced by the amount of prior experience women have had caring for young. While the role of hormones in mediating maternal behavior in women is still unclear, initial results indicate they may facilitate responsiveness during the puerperium.     

Cerebral blood flow in the subgenual anterior cingulate cortex and modulation of the mood-regulatory networks in a successful rTMS treatment for major depressive disorder

The subgenual anterior cingulate cortex (Cg25) has been reported to be a node of mood-regulatory networks. Using a responder and a non-responder of repetitive transcranial magnetic stimulation (rTMS) for depression, we examined pre/post-treatment cerebral blood flow (CBF) in the Cg25 and treatment-related CBF changes in cortical/subcortical regions. In the responder, pre-treatment Cg25 perfusion was higher and was decreased after treatment, in addition, CBF was increased in the frontal and parietal regions and decreased in the hippocampus and basal ganglia. Our results suggest that rTMS treatment response may be related to pre-treatment Cg25 activity and modulation of the Cg25 and mood-regulatory networks.     

Decreased brain docosahexaenoic acid content produces neurobiological effects associated with depression: Interactions with reproductive status in female rats

Decreased tissue levels of docosahexaenoic acid (DHA; 22:6n-3) are implicated in the etiologies of non-puerperal and postpartum depression. With the aim of determining neurobiological sequelae of decreased brain DHA content, this study examined the effects of a loss of brain DHA content and concurrent reproductive status in adult female Long-Evans rats. An α-linolenic acid-deficient diet and breeding protocols were used to produce virgin and parous female rats with cortical phospholipid DHA levels 23–26% lower than virgin and parous rats fed a control diet containing adequate α-linolenic acid. Parous dams were tested/euthanized at weaning (postnatal day 20) of the second litter; virgin females, during diestrus. Decreased brain DHA was associated with decreased hippocampal BDNF gene expression and increased relative corticosterone response to an intense stressor, regardless of reproductive status. In virgin females with decreased brain DHA, serotonin content and turnover in frontal cortex were decreased compared to virgin females with normal brain DHA. In parous dams with decreased brain DHA, the density of 5-HT_1A receptors in the hippocampus was increased, corticosterone response to an intense stressor was increased, and the latency to immobility in the forced swim test was decreased compared to parous dams with normal DHA. These findings demonstrate neurobiological alterations attributable to decreased brain DHA or an interaction of parous status and brain DHA level. Furthermore, the data are consistent with findings in depressed humans, and thus support a role for DHA as a factor in the etiologies of depressive illnesses, particularly postpartum depression.     

Intranasal Insulin Ameliorates Cerebral Hypometabolism,Neuronal Loss,and Astrogliosis in Streptozotocin-Induced Alzheimer’s Rat Model

Intracerebroventricular injection of streptozotocin (ICV-STZ) in rodents leads to cognitive impairments and several pathological changes like Alzheimer’s disease (AD). However, there is hardly any research about the effect of ICV-STZ on regional cerebral glucose metabolism in rodents. Previous studies have demonstrated that intranasal insulin improves cognition in AD patients. However, the underlying mechanism remains elusive. Here, we treated the ICV-STZ rats with daily intranasal delivery of insulin (2 U/day) for 6 consecutive weeks, then monitored ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) uptake using a high-resolution small-animal positron emission tomography (microPET) and studied the expression of neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) using immunohistochemical staining. We observed that ¹⁸F-FDG uptake decreased significantly at the prefrontal cortex, cingulate cortex, striatum, hippocampus, and entorhinal cortex in ICV-STZ rats as compared with the control rats. Intranasal insulin restores the cerebral glucose metabolism in prefrontal and cingulate cortex and attenuates astroglia activation and neuronal loss in the hippocampus of ICV-STZ rats. These findings provide the mechanistic basis for treating AD patients with intranasal insulin.     

Offspring-exposure reduces depressive-like behaviour in the parturient female rat

In women, breastfeeding generally results in reductions in anxiety and increased positive mood. However, approximately 10-15% of women experience depressed mood and increased anxiety during the first year postpartum. Recent research has demonstrated that offspring-exposure is important for the reduction in behaviours related to depression and anxiety in the mother. It remains to be determined whether these effects are due to factors related to pregnancy and/or pup-exposure, are associated with the degree of maternal behaviour by the mother towards offspring, or persist after weaning. To address these questions the present study used four groups of female rats; primiparous, nulliparous, primip-no-pups (primiparous females with pups permanently removed), and sensitized females. Depressive- and anxiety-like behaviours were assessed 1 week after weaning/pup-exposure (4 weeks after birth for primip-no-pups animals) using the forced swim test for measures of depressive-like behaviour, and the open field test and elevated plus maze for measure of anxiety-like behaviour. Results demonstrate that primiparous females without pup-exposure have increased depressive-like, but not anxiety-like, behaviour compared to primiparous and sensitized females. In addition, kyphotic nursing by primiparous mothers was negatively related to behavioural measures of depression and anxiety. From this work it is clear that pup-exposure is important for reductions in depressive-like behaviour in parturient females. Further research is needed to determine the extent of these changes and the neural and hormonal correlates of these events.     

Response of rat cerebral cortical astrocytes to freeze- or cobalt-induced injury: an immunocytochemical and gap-FRAP study

Astrocytic response in the immediate vicinity of freeze- and cobalt-induced lesions has been examined at the light and ultrastructural level. However, the temporal and spatial distribution of astrocytic reactivity throughout the rat cerebral cortex, using glial fibrillary acidic protein (GFAP) immunolabeling, has not been examined. The first purpose of this study was to establish the chronological distribution of astrocytic reactivity, as measured by changes in GFAP immunoreactivity, following freeze- or cobalt-induced injury to the rat cerebral cortex. Cobalt metal also has been proposed to have a direct effect on astrocytes and has been shown to stimulate in vitro astrocytes to become reactive. The second purpose of this report was to determine if cobalt had an effect on in vitro astrocytic gap junctional dye coupling as measured by fluorescence recovery after laser-photobleaching (gap-FRAP). Although the chronological development of the increased GFAP immunoreactivity was different for the freeze- and cobalt-induced lesions, astrocytes initially showed an increase in GFAP immunoreactivity in the region surrounding these lesions. This initial response was followed by a spread of increased GFAP immunoreactivity throughout certain regions of the ipsilateral cerebral hemisphere and then by a restriction of the increased immunolabeling to the lesion site. Cobalt also had a direct effect on in vitro astrocytes as demonstrated by the inhibition of astrocytic gap junctional dye coupling. Based on gap-FRAP analysis, cobalt significantly blocked fluorescence recovery (2.5%) as compared to the fluorescence recovery in control astrocytes (26%). It is proposed that the initial increase in GFAP immunoreactivity may be due to decreased gap junctional activity.