Neonatal Prefrontal Cortex Research Articles

SLITRK1-mediated noradrenergic projection suppression in the neonatal prefrontal cortex

SLITRK1 is an obsessive-compulsive disorder spectrum-disorders-associated gene that encodes a neuronal transmembrane protein. Here we show that SLITRK1 suppresses noradrenergic projections in the neonatal prefrontal cortex, and SLITRK1 functions are impaired by SLITRK1 mutations in patients with schizophrenia (S330A, a revertant of Homo sapiens-specific residue) and bipolar disorder (A444S). Slitrk1-KO newborns exhibit abnormal vocalizations, and their prefrontal cortices show excessive noradrenergic neurites and reduced Semaphorin3A expression, which suppresses noradrenergic neurite outgrowth in vitro. Slitrk1 can bind Dynamin1 and L1 family proteins (Neurofascin and L1CAM), as well as suppress Semaphorin3A-induced endocytosis. Neurofascin-binding kinetics is altered in S330A and A444S mutations. Consistent with the increased obsessive-compulsive disorder prevalence in males in childhood, the prefrontal cortex of male Slitrk1-KO newborns show increased noradrenaline levels, and serotonergic varicosity size. This study further elucidates the role of noradrenaline in controlling the development of the obsessive-compulsive disorder-related neural circuit.

Knock-Down of Hippocampal DISC1 in Immune-Challenged Mice Impairs the Prefrontal–Hippocampal Coupling and the Cognitive Performance Throughout Development

Disrupted-in-schizophrenia 1 (DISC1) gene represents an intracellular hub of developmental processes. When combined with early environmental stressors, such as maternal immune activation, but not in the absence of thereof, whole-brain DISC1 knock-down leads to memory and executive deficits as result of impaired prefrontal–hippocampal communication throughout development. While synaptic dysfunction in neonatal prefrontal cortex (PFC) has been recently identified as one source of abnormal long-range coupling, the contribution of hippocampus (HP) is still unknown. Here, we aim to fill this knowledge gap by combining in vivo electrophysiology and optogenetics with morphological and behavioral assessment of immune-challenged mice with DISC1 knock-down either in the whole brain (GE) or restricted to pyramidal neurons in hippocampal CA1 area (GHPE). We found abnormal network activity, sharp-waves, and neuronal firing in CA1 that complement the deficits in upper layer of PFC. Moreover, optogenetic activating CA1 pyramidal neurons fails to activate the prefrontal local circuits. These deficits that persist till prejuvenile age relate to dendrite sparsification and loss of spines of CA1 pyramidal neurons. As a long-term consequence, DISC1 knock-down in HP leads to poorer recognition memory at prejuvenile age. Thus, DISC1-controlled developmental processes in HP in immune-challenged mice are critical for circuit function and cognitive behavior.

The Thalamus Regulates Retinoic Acid Signaling and Development of Parvalbumin Interneurons in Postnatal Mouse Prefrontal Cortex.

GABAergic inhibitory neurons in the prefrontal cortex (PFC) play crucial roles in higher cognitive functions. Despite the link between aberrant development of PFC interneurons and a number of psychiatric disorders, mechanisms underlying the development of these neurons are poorly understood. Here we show that the retinoic acid (RA)-degrading enzyme CYP26B1 (cytochrome P450 family 26, subfamily B, member 1) is transiently expressed in the mouse frontal cortex during postnatal development, and that medial ganglionic eminence (MGE)-derived interneurons, particularly in parvalbumin (PV)-expressing neurons, are the main cell type that has active RA signaling during this period. We found that frontal cortex-specific Cyp26b1 knock-out mice had an increased density of PV-expressing, but not somatostatin-expressing, interneurons in medial PFC, indicating a novel role of RA signaling in controlling PV neuron development. The initiation of Cyp26b1 expression in neonatal PFC coincides with the establishment of connections between the thalamus and the PFC. We found that these connections are required for the postnatal expression of Cyp26b1 in medial PFC. In addition to this region-specific role in postnatal PFC that regulates RA signaling and PV neuron development, the thalamocortical connectivity had an earlier role in controlling radial dispersion of MGE-derived interneurons throughout embryonic neocortex. In summary, our results suggest that the thalamus plays multiple, temporally separate roles in interneuron development in the PFC.

Glycine release from astrocytes via functional reversal of GlyT1.

It was previously reported that functional glycine receptors were expressed in neonatal prefrontal cortex; however, the glycine-releasing cells were unknown. We hypothesized that astrocytes might be a major glycine source, and examined the glycine release properties of astrocytes. We also hypothesized that dopamine (DA) might be a trigger for the astrocytic glycine release, as numerous DA terminals localize in the cortex. We combined two different methods to confirm the glycine release from astrocytes. Firstly, we analyzed the supernatant of astrocytes by amino acid analyzer after DA stimulation, and detect significant glycine peak. Furthermore, we utilized a patch-clamp biosensor method to confirm the glycine release from astrocytes by using GlyRα1 and Glyβ-expressing HEK293T cells, and detected significant glycine-evoked current upon DA stimulation. Thus, we clearly demonstrated that DA induces glycine release from astrocytes. Surprisingly, DA caused a functional reversal of astrocytic glycine transporter 1, an astrocytic type of glycine transporter, causing astrocytes to release glycine. Hence, astrocytes transduce pre-synaptic DA signals to glycine signals through a reversal of astrocytic glycine transporter 1 to regulate neuronal excitability. Cover Image for this issue: doi: 10.1111/jnc.13785.

Na+, K+-ATPase dysfunction causes cerebrovascular endothelial cell degeneration in rat prefrontal cortex slice cultures

Cerebrovascular endothelial cell dysfunction resulting in imbalance of cerebral blood flow contributes to the onset of psychiatric disorders such as depression, schizophrenia and bipolar disorder. Although decrease in Na+, K+-ATPase activity has been reported in the patients with schizophrenia and bipolar disorder, the contribution of Na+, K+-ATPase to endothelial cell dysfunction remains poorly understood. Here, by using rat neonatal prefrontal cortex slice cultures, we demonstrated that pharmacological inhibition of Na+, K+-ATPase by ouabain induced endothelial cell injury. Treatment with ouabain significantly decreased immunoreactive area of rat endothelial cell antigen-1 (RECA-1), a marker of endothelial cells, in a time-dependent manner. Ouabain also decreased Bcl-2/Bax ratio and phosphorylation level of glycogen synthase kinase 3β (GSK3β) (Ser9), which were prevented by lithium carbonate. On the other hand, ouabain-induced endothelial cell injury was exacerbated by concomitant treatment with LY294002, an inhibitor of phosphoinositide 3- (PI3-) kinase. We also found that xestospongin C, an inhibitor of inositol triphosphate (IP3) receptor, but not SEA0400, an inhibitor of Na+, Ca2+ exchanger (NCX), protected endothelial cells from cytotoxicity of ouabain. These results suggest that cerebrovascular endothelial cell degeneration induced by Na+, K+-ATPase inhibition resulting in Ca2+ release from endoplasmic reticulum (ER) and activation of GSK3β signaling underlies pathogenesis of these psychiatric disorders.

Thalamic and Entorhinal Network Activity Differently Modulates the Functional Development of Prefrontal-Hippocampal Interactions.

Memories are encoded by finely tuned interactions within large-scale neuronal networks. This cognitive performance is not inherited, but progressively matures in relationship with the establishment of long-range coupling in the immature brain. The hippocampus initiates and unidirectionally drives the oscillatory entrainment of neonatal prefrontal cortex, yet feedback interactions that precisely control this early communication are still unresolved. Here, we identified distinct roles of entorhinal cortex and ventral midline thalamus for the functional development of prefrontal-hippocampal interactions. While entorhinal oscillations modulate the hippocampal activity by timing the neuronal firing via monosynaptic afferents, thalamic nuclei act as a relay station routing prefrontal activation back to hippocampus. Understanding the mechanisms of network maturation represents the prerequisite for assessing circuit dysfunction in neurodevelopmental disorders.

Dendritic morphology changes in neurons from the ventral hippocampus, amygdala and nucleus accumbens in rats with neonatal lesions into the prefrontal cortex.

Neonatal prefrontal cortex (nPFC) lesions in rats could be a potential animal model to study the early neurodevelopmental abnormalities associated with the behavioral and morphological brain changes observed in schizophrenia. Morphological alterations in pyramidal neurons from the ventral hippocampus (VH) have been observed in post-mortem schizophrenic brains, mainly because of decreased dendritic arbor and spine density. We assessed the effects of nPFC-lesions on the dendritic morphology of neurons from the VH, basolateral-amygdala (BLA) and the nucleus accumbens (NAcc) in rats. nPFC lesions were made on postnatal day 7 (PD7), after dendritic morphology was studied by the Golgi-Cox stain procedure followed by Sholl analysis at PD35 (prepubertal) and PD60 (adult) ages. We also evaluated the effects of PFC-lesions on locomotor activity caused by a novel environment. Adult animals with nPFC lesions showed a decreased spine density in pyramidal neurons from the VH and in medium spiny cells from the NAcc. An increased locomotion was observed in a novel environment for adult animals with a PFC-lesion. Our results indicate that PFC-lesions alter the neuronal dendrite morphology of the NAcc and the VH, suggesting a disconnection between these limbic structures. The locomotion paradigms suggest that dopaminergic transmission is altered in the PFC lesion model. This could help to understand the consequences of an earlier PFC dysfunction in schizophrenia. To evaluate possible dendritic changes in neonatal prefrontal cortex lesions in schizophrenia-related regions including nucleus accumbens, ventral hippocampus and basolateral amygdala, we used the Golgi-Cox stain samples at PD35 and PD70. Our results suggest that neonatal prefrontal cortex damage alters dendritic parameters in limbic regions, and this has potential implications for schizophrenia.

17β-estradiol attenuates ketamine-induced neuroapoptosis and persistent cognitive deficits in the developing brain

Previous studies have demonstrated that the commonly used anesthetic ketamine can induce widespread neuroapoptosis in the neonatal brain and can cause persistent cognitive impairments as the animal matures. Therefore, searching for adjunctive neuroprotective strategies that inhibit ketamine-induced neuroapoptosis and persistent cognitive impairments is highly warranted. The primary goal of this study was to investigate the protective effect of 17β-estradiol against ketamine-induced neuroapoptosis and persistent cognitive impairments in adult rats. Starting from postnatal day 7, Sprague-Dawley male rat pups were given a daily administration of ketamine (75mg/kg, i.p.) or 17β-estradiol (600μg/kg, s.c.) in combination with ketamine (75mg/kg, i.p.). The animals were treated for three consecutive days. 24h after the last injection, the rats were decapitated, and the prefrontal cortex (PFC) was isolated to detect neuroapoptosis by cleaved caspase-3 immunohistochemistry and by using the TUNEL assay. The neuroactive steroid 17β-estradiol was quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The protein levels of BDNF and pAkt were measured by western blot analysis. At two months of age (60 days), the learning and memory abilities were tested using the Morris water maze. The results showed that ketamine triggered significant neuroapoptosis in the neonatal PFC accompanied by the downregulation of 17β-estradiol, BDNF and pAkt. The co-administration of 17β-estradiol with ketamine attenuated these changes. Moreover, 17β-estradiol significantly reversed the learning and memory deficits observed at 60 days of age. In brief, our present data demonstrate that 17β-estradiol attenuates ketamine-induced neuroapoptosis and reverses long-term cognitive deficits in developing rats and thus may be a potential therapeutic and neuroprotective method for the treatment of neurodevelopmental disorders.This article is part of a Special Issue entitled SI: Brain and Memory.

Pharmacological characterisation of ligand- and voltage-gated ion channels expressed in human iPSC-derived forebrain neurons

Genetic causes, or predisposition, are increasingly accepted to be part of the ethiopathogenesis of many neuropsychiatric diseases. While genes can be studied in any type of cells, their physiological function in human brain cells is difficult to evaluate, particularly in living subjects. As a first step towards the characterisation of human inducible pluripotent stem cell (iPSC)-derived neurons from autism spectrum disorder (ASD) patients, we used gene expression and functional studies to define the regional identity of the typical forebrain differentiation, demonstrate expression patterns of genes of interest in ASD and understand the properties of 'control' iPSC-derived neurons (iCell-Neurons™), with a focus on receptors and ion channels that play a central role in synaptic physio-pathology. The gene expression profile of the iCell-Neurons™ closely resembled that observed in neonatal prefrontal cortex tissues. Functional studies, performed mainly using calcium flux assays, demonstrated the presence of ionotropic glutamate (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate) and gamma-aminobutyric acid type A receptors. Voltage-gated sodium and calcium channels were also identified using similar techniques. Overall, the results reported here suggest that iCell-Neurons™ are a good cellular model of a relatively immature forebrain human neuron population that can be used both as a control in comparison to patients cells, and as host cells in which mutations, insertions and deletions can be used in order to study the molecular mechanisms of ASD and other neurological disorders in an isogenic cellular background.

Hypoxia-Ischemia Disrupts Directed Interactions within Neonatal Prefrontal-Hippocampal Networks

Due to improved survival rates and outcome of human infants experiencing a hypoxic-ischemic episode, cognitive dysfunctions have become prominent. They might result from abnormal communication within prefrontal-hippocampal networks, as synchrony and directed interactions between the prefrontal cortex and hippocampus account for mnemonic and executive performance. Here, we elucidate the structural and functional impact of hypoxic-ischemic events on developing prefrontal-hippocampal networks in an immature rat model of injury. The magnitude of infarction, cell loss and astrogliosis revealed that an early hypoxic-ischemic episode had either a severe or a mild/moderate outcome. Without affecting the gross morphology, hypoxia-ischemia with mild/moderate outcome diminished prefrontal neuronal firing and gamma network entrainment. This dysfunction resulted from decreased coupling synchrony within prefrontal-hippocampal networks and disruption of hippocampal theta drive. Thus, early hypoxia-ischemia may alter the functional maturation of neuronal networks involved in cognitive processing by disturbing the communication between the neonatal prefrontal cortex and hippocampus.

Oscillatory coupling within neonatal prefrontal–hippocampal networks is independent of selective removal of GABAergic neurons in the hippocampus

GABAergic neurons have been proposed to control oscillatory entrainment and cognitive processing in prefrontal–hippocampal networks. Co-activation of these networks emerges already during neonatal development, with hippocampal theta bursts driving prefrontal oscillations via axonal projections. The cellular substrate of neonatal prefrontal–hippocampal communication and in particular, the role of GABAergic neurons, is still unknown. Here, we used saporin-conjugated anti-vesicular GABA transporter antibodies to cause selective immunotoxic lesion of GABAergic neurons in the CA1 area of the hippocampus during the first postnatal week. Without affecting the somatic development of rat pups, the lesion impaired the generation of hippocampal sharp waves, but not of theta bursts during neonatal development. Moreover, the oscillatory entrainment and firing of neonatal prefrontal cortex as well as the early prefrontal–hippocampal synchrony were largely independent of GABAergic neurotransmission in the hippocampus. Thus, hippocampal interneurons are critical elements for the ontogeny of hippocampal sharp waves, but seem to not control the directed oscillatory coupling between the neonatal prefrontal cortex and hippocampus.

Comparative behavioral changes in postpubertal rats after neonatal excitotoxic lesions of the ventral hippocampus and the prefrontal cortex

The neonatal ventral hippocampal (nVH) and the neonatal prefrontal cortex (nPFC) lesions in rats have been used as models to test the hypothesis that early neurodevelopmental abnormalities lead to behavioral changes putatively linked to schizophrenia. We investigated the role of the nVH and the nPFC lesions on behavioral characteristics related to locomotor behaviors, social interaction, and grooming. Bilateral ibotenic acid lesions of the VH, the PFC, or both were made in neonatal Sprague-Dawley rats (postnatal day 7, P7) and their behaviors studied at P35 and P60. No significant differences in any of the behaviors were observed between sham animals and rats with ibotenic acid lesions at P35. Postpubertally (at P60), the spontaneous locomotor activity of nVH-lesioned rats was significantly enhanced compared to the sham controls; however, this hyperactivity was reversed by nVH and nPFC double lesions. Neonatal PFC lesion alone did not alter spontaneous activity, although a trend of increased activity was observed. The duration of grooming was significantly decreased in rats with neonatal lesions of the VH. Similar to the data on locomotion, nVH plus nPFC lesion normalized the grooming behavior. Lesion of the PFC alone was without any significant effect on grooming behavior. Neonatal VH-lesioned animals spent less time in active social interaction, and this effect persisted even in nVH plus nPFC-lesioned animals. By itself, nPFC lesion did not alter social behavior. These data suggest that subtle developmental aberrations within PFC caused by nVH lesions, rather than the lesion of PFC itself, may contribute to some of the behavioral changes seen in the nVH-lesioned rats.

Serotonin Induces Tonic Firing in Layer V Pyramidal Neurons of Rat Prefrontal Cortex during Postnatal Development

The effects of serotonin (5-HT) on neuronal activity were examined during postnatal development in layer V pyramidal neurons of the rat prefrontal cortex (PFC) in vitro. Whole-cell patch-clamp recordings were made in slices obtained from rats aged between postnatal day (P) 6 and P31. In P14 or younger neurons, bath application of 5-HT (10 microM) induced a large depolarization followed by tonic firing at 2-5 Hz. The excitatory effects of 5-HT decreased rapidly after P14, so that by P21, 5-HT produced a small depolarization or hyperpolarization without cell firing. The excitatory effects of 5-HT at younger ages were attributed to 5-HT2A receptors because the effects were mimicked by the 5-HT2 agonist alpha-methyl-5-HT but not by the 5-HT3 agonist 1-(m-chlorophenyl)-biguanide, nor by the 5-HT2B/2C agonist 1-(3-chlorophenyl)piperazine, and were blocked by the 5-HT2A antagonists ketanserin and alpha-phenyl-1-(2-phenylethyl)-4-piperidinemethanol. The excitatory responses persisted in 0 [Ca2+]o and high [Mg2+]o in the presence of TTX or blockers of ionotropic glutamate receptors, suggesting that the effects were mediated essentially by postsynaptic mechanisms. The responses to 5-HT involve a reduction of K+ conductance and an enhancement of the hyperpolarization-activated Na+/K+ current. The developmental decline of 5-HT-induced excitatory effects was associated with a downregulation of 5-HT2A receptor function and a decrease in the input resistance during early life. These results suggest that 5-HT is an important regulator of neuronal activity in the neonatal PFC and may play a role in activity-dependent developmental processes.

Enhanced hippocampal-mediated HPA response to stress in schizophrenia

The medial prefrontal cortex modulates the nucleus accumbens dopamine response to stress and has been implicated in feedback regulation of hypothalamic–pituitary–adrenal axis activation by stress. Here we report on the effects of bilateral neonatal (postnatal day 7) ibotenate-induced lesions to the medial prefrontal cortex on nucleus accumbens dopamine and neuroendocrine function in adult rats. Voltammetry was used to monitor the dopamine response to each of five, once-daily exposures to tail-pinch stress whereas alterations in neuroendocrine function were determined from the plasma corticosterone response to a single 20-min episode of restraint stress. Potential lesion-induced deficits in sensory-motor gating were assessed by measuring prepulse inhibition of the acoustic startle response before and after repeated stress. Our data show that each daily stress episode elicited larger and longer-lasting dopamine increases in prefrontal cortex-lesioned animals than in sham-lesioned controls. Furthermore, greater stress-induced elevations in plasma corticosterone were seen in lesioned animals than in their sham-lesioned counterparts. However, while repeated stress potentiated startle responses in animals of both groups, there was no effect of lesion on the amplitude or on prepulse inhibition of the startle response.Together, these findings indicate that neonatal prefrontal cortex damage can lead to changes in mesolimbic dopamine and neuroendocrine function during adulthood. They also add to a growing body of experimental and clinical evidence implicating abnormal prefrontal cortex neuronal development in the pathophysiology of schizophrenia and other disorders linked to central dopamine dysfunction.

Enhanced nucleus accumbens dopamine and plasma corticosterone stress responses in adult rats with neonatal excitotoxic lesions to the medial prefrontal cortex

The medial prefrontal cortex modulates the nucleus accumbens dopamine response to stress and has been implicated in feedback regulation of hypothalamic–pituitary–adrenal axis activation by stress. Here we report on the effects of bilateral neonatal (postnatal day 7) ibotenate-induced lesions to the medial prefrontal cortex on nucleus accumbens dopamine and neuroendocrine function in adult rats. Voltammetry was used to monitor the dopamine response to each of five, once-daily exposures to tail-pinch stress whereas alterations in neuroendocrine function were determined from the plasma corticosterone response to a single 20-min episode of restraint stress. Potential lesion-induced deficits in sensory-motor gating were assessed by measuring prepulse inhibition of the acoustic startle response before and after repeated stress. Our data show that each daily stress episode elicited larger and longer-lasting dopamine increases in prefrontal cortex-lesioned animals than in sham-lesioned controls. Furthermore, greater stress-induced elevations in plasma corticosterone were seen in lesioned animals than in their sham-lesioned counterparts. However, while repeated stress potentiated startle responses in animals of both groups, there was no effect of lesion on the amplitude or on prepulse inhibition of the startle response. Together, these findings indicate that neonatal prefrontal cortex damage can lead to changes in mesolimbic dopamine and neuroendocrine function during adulthood. They also add to a growing body of experimental and clinical evidence implicating abnormal prefrontal cortex neuronal development in the pathophysiology of schizophrenia and other disorders linked to central dopamine dysfunction.

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.

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.