Longitudinal Biochemical and Behavioral Alterations in a Gyrencephalic Model of Blast-Related Mild Traumatic Brain Injury.

Blast-related traumatic brain injury (bTBI) is a major cause of neurological disorders in the U.S. military that can adversely impact some civilian populations as well and can lead to lifelong deficits and diminished quality of life. Among these types of injuries, the long-term sequelae are poorly understood because of variability in intensity and number of the blast exposure, as well as the range of subsequent symptoms that can overlap with those resulting from other traumatic events (e.g., post-traumatic stress disorder). Despite the valuable insights that rodent models have provided, there is a growing interest in using injury models using species with neuroanatomical features that more closely resemble the human brain. With this purpose, we established a gyrencephalic model of blast injury in ferrets, which underwent blast exposure applying conditions that closely mimic those associated with primary blast injuries to warfighters. In this study, we evaluated brain biochemical, microstructural, and behavioral profiles after blast exposure using in vivo longitudinal magnetic resonance imaging, histology, and behavioral assessments. In ferrets subjected to blast, the following alterations were found: 1) heightened impulsivity in decision making associated with pre-frontal cortex/amygdalar axis dysfunction; 2) transiently increased glutamate levels that are consistent with earlier findings during subacute stages post-TBI and may be involved in concomitant behavioral deficits; 3) abnormally high brain N-acetylaspartate levels that potentially reveal disrupted lipid synthesis and/or energy metabolism; and 4) dysfunction of pre-frontal cortex/auditory cortex signaling cascades that may reflect similar perturbations underlying secondary psychiatric disorders observed in warfighters after blast exposure.

The effect of developmental alcohol exposure on multisensory integration is larger in deeper cortical layers.

Fetal Alcohol Spectrum Disorders (FASD) are one of the most common causes of mental disability in the world. Despite efforts to increase public awareness of the risks of drinking during pregnancy, epidemiological studies indicate a prevalence of 1-6% in all births. There is growing evidence that deficits in sensory processing may contribute to social problems observed in FASD. Multisensory (MS) integration occurs when a combination of inputs from two sensory modalities leads to enhancement or suppression of neuronal firing. MS enhancement is usually linked to processes that facilitate cognition and reaction time, whereas MS suppression has been linked to filtering unwanted sensory information. The rostral portion of the posterior parietal cortex (PPr) of the ferret is an area that shows robust visual-tactile integration and displays both MS enhancement and suppression. Recently, our lab demonstrated that ferrets exposed to alcohol during the "third trimester equivalent" of human gestation show less MS enhancement and more MS suppression in PPr than controls. Here we complement these findings by comparing in vivo electrophysiological recordings from channels located in shallow and deep cortical layers. We observed that while the effects of alcohol (less MS enhancement and more MS suppression) were found in all layers, the magnitude of these effects were more pronounced in putative layers V-VI. These findings extend our knowledge on the sensory deficits of FASD.

Development of multisensory processing in ferret parietal cortex.

It is well known that the nervous system adjusts itself to its environment during development. Although a great deal of effort has been directed towards understanding the developmental processes of the individual sensory systems (e.g., vision, hearing, etc.), only one major study has examined the maturation of multisensory processing in cortical neurons. Therefore, the present investigation sought to evaluate multisensory development in a different cortical region and species. Using multiple single-unit recordings in anaesthetised ferrets (n = 18) of different ages (from postnatal day 80 to 300), we studied the responses of neurons from the rostral posterior parietal (PPr) area to presentations of visual, tactile and combined visual-tactile stimulation. The results showed that multisensory neurons were infrequent at the youngest ages (pre-pubertal) and progressively increased through the later ages. Significant response changes that result from multisensory stimulation (defined as multisensory integration [MSI]) were observed in post-pubertal adolescent animals, and the magnitude of these integrated responses also increased across this age group. Furthermore, non-significant multisensory response changes were progressively increased in adolescent animals. Collectively, at the population level, MSI was observed to shift from primarily suppressive levels in infants to increasingly higher levels in later stages. These data indicate that, like the unisensory systems from which it is derived, multisensory processing shows developmental changes whose specific time course may be regionally and species-dependent.

Combined exposure to alcohol and cannabis during development: Mechanisms and outcomes.

Exposure to substances of abuse during pregnancy can have long-lasting effects on offspring. Alcohol is one of the most widely used substances of abuse that leads to the most severe consequences. Recent studies in the United States, Canada, and the United Kingdom showed that between 1% and 7% of all children exhibit signs and symptoms of fetal alcohol spectrum disorder (FASD). Despite preventive campaigns, the rate of children with FASD has not decreased during recent decades. Alcohol consumption often accompanies exposure to such drugs as tobacco, cocaine, opioids, and cannabis. These interactions can be synergistic and exacerbate the deleterious consequences of developmental alcohol exposure. The present review focuses on interactions between alcohol and cannabis exposure and the potential consequences of these interactions.

Effects of developmental alcohol exposure on cortical multisensory integration.

Fetal alcohol spectrum disorder (FASD) is one of the most common causes of mental disabilities in the world with a prevalence of 1%-6% of all births. Sensory processing deficits and cognitive problems are a major feature in this condition. Because developmental alcohol exposure can impair neuronal plasticity, and neuronal plasticity is crucial for the establishment of neuronal circuits in sensory areas, we predicted that exposure to alcohol during the third trimester equivalent of human gestation would disrupt the development of multisensory integration (MSI) in the rostral portion of the posterior parietal cortex (PPr), an integrative visual-tactile area. We conducted in vivo electrophysiology in 17 ferrets from four groups (saline/alcohol; infancy/adolescence). A total of 1157 neurons were recorded after visual, tactile and combined visual-tactile stimulation. A multisensory (MS) enhancement or suppression is characterized by a significantly increased or decreased number of elicited spikes after combined visual-tactile stimulation compared to the strongest unimodal (visual or tactile) response. At the neuronal level, those in infant animals were more prone to show MS suppression whereas adolescents were more prone to show MS enhancement. Although alcohol-treated animals showed similar developmental changes between infancy and adolescence, they always 'lagged behind' controls showing more MS suppression and less enhancement. Our findings suggest that alcohol exposure during the last months of human gestation would stunt the development of MSI, which could underlie sensory problems seen in FASD.

Alterations in motor functional connectivity in Neonatal Hypoxic Ischemic Encephalopathy.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) is the result of global hypoxic-ischemic brain injury in neonates due to asphyxia during birth and is one of the most common causes of severe, long-term neurologic deficits in children. Methods: Resting state fMRI (rs-fMRI) was used to assess potential functional disruptions in the primary and association motor areas in HIE neonates (n = 16) compared to healthy controls (n = 11). RESULTS: Results demonstrate reduced intra-hemispheric resting state functional connectivity (rs-FC) between primary motor regions (upper extremity and facial motor regions) as well as reduced inter-hemispheric rs-FC in the HIE group. In addition, HIE neonates demonstrated increased rs-FC between motor regions and frontal, temporal and parietal cortices but decreased rs-FC with the cerebellum. DISCUSSION: These preliminary results provide initial evidence for the disruption of functional communication with the motor network in neonates with HIE. Further studies are necessary to both validate these findings in a larger dataset as well as to determine if rs-fMRI measurements collected at birth may have the potential to serve as a prognostic marker in addition to the traditional combination of clinical measurements and conventional MRI.

Phosphorylation of CREB at Serine 142 and 143 Is Essential for Visual Cortex Plasticity.

The transcription factor cAMP response element-binding protein (CREB) is involved in a myriad of cellular functions in the central nervous system. For instance, the role of CREB via phosphorylation at the amino-acid residue Serine (Ser)133 in expressing plasticity-related genes and activity-dependent neuronal plasticity processes has been extensively demonstrated. However, much less is known about the role of CREB phosphorylation at Ser142 and Ser143. Here, we employed a viral vector containing a dominant negative form of CREB, with serine-to-alanine mutations at residue 142 and 143 to specifically block phosphorylation at both sites. We then transfected this vector into primary neurons in vitro or intracortically injected it into mice in vivo, to test whether these phosphorylation events were important for activity-dependent plasticity. We demonstrated by immunohistochemistry of cortical neuronal cultures that the expression of Arc, a known plasticity-related gene, requires triple phosphorylation of CREB at Ser133, Ser142, and Ser143. Moreover, we recorded visually-evoked field potentials in awake mice before and after a 7-d period of monocular deprivation (MD) to show that, in addition to CREB phosphorylation at Ser133, ocular dominance plasticity (ODP) in the visual cortex also requires CREB phosphorylation at Ser142/143. Our findings suggest that Ser142/143 phosphorylation is an additional post-translational modification of CREB that triggers the expression of specific target genes and activity-dependent neuronal plasticity processes.

Vinpocetine, cognition, and epilepsy.

OBJECTIVE: Vinpocetine has been shown to enhance memory in animal models, with possible cognitive benefit in humans. The present study sought to demonstrate if vinpocetine can enhance cognition in healthy volunteers or patients with epilepsy. In addition, we compare blood levels of vinpocetine and its active metabolite (apovincaminic acid; AVA) in humans and animals to further characterize factors related to possible therapeutic benefit. METHODS: The cognitive effects of vinpocetine were assessed in healthy adult volunteers (n = 8) using a double-blind, randomized, crossover design at single doses (placebo, 10, 20, and 60 mg oral). Cognitive effects of vinpocetine in patients with focal epilepsy (n = 8) were tested using a double-blind, randomized, crossover design at single doses (placebo, 20 mg oral) followed by one-month open label at 20 mg oral three times a day. The neuropsychological battery included both computerized and non-computerized tests. Levels of vinpocetine and AVA in the human studies were compared to levels in 45 mice across time dosed at 5-20 mg/kg intraperitoneal of vinpocetine. RESULTS: No significant cognitive benefits were seen in healthy volunteers or patients with epilepsy. No appreciable side effects occurred. Vinpocetine and AVA levels were lower in humans than animals. CONCLUSIONS: Vinpocetine was well tolerated, but was not associated with positive cognitive effects. However, blood levels obtained in humans were substantially less than levels in animals obtained from dosages known to be effective in one model. This suggests that higher dosages are needed in humans to assess vinpocetine's cognitive efficacy.

Mercury, lead, and cadmium exposure via red blood cell transfusions in preterm infants.

BACKGROUND: Mercury, lead, and cadmium are developmental neurotoxicants. We predict that preterm newborns requiring packed red blood cell (PRBC) transfusions may be exposed to neurotoxic doses. We explored the relationship between donor concentration, number of donors, number of transfusions and mercury, lead and cadmium exposure. METHODS: Single-donor PRBCs were analyzed for mercury, lead and cadmium concentration. Dose per transfusion was calculated and compared to intravenous reference doses (IVRfDs). Linear regression analyses were performed to correlate donor and infant exposure. RESULTS: Thirty-six infants received 268 transfusions from 94 donors. Number of donors and transfusions were significantly correlated with birthweight and gestational age. All three metals were detected in ≥95% of donor PRBCs. Number of donors was significantly associated with cumulative dose, and there was a significant correlation between mercury and lead doses/transfusion. IVRfDs were exceeded for mercury and lead in 8.6% and 38% of transfusions, respectively. None exceeded the IVRfD for cadmium. For lead, infants exposed to three donors had more transfusions exceeding IVRfD than those exposed to 1-2 donors. CONCLUSIONS: Preterm infants are exposed to heavy metals via transfusions. Doses exceeded the IVRfDs for mercury and lead. Cadmium did not pose a risk. Prescreening donor blood could reduce exposure risk.

Effect of Postmortem Interval and Years in Storage on RNA Quality of Tissue at a Repository of the NIH NeuroBioBank.

Brain tissue from 1068 donors was analyzed for RNA quality as a function of postmortem interval (PMI) and years in storage. Approximately 83% of the cortical and cerebellar samples had an RNA integrity number (RIN) of 6 or greater, indicating their likely suitability for real-time quantitative polymerase chain reaction research. The average RIN value was independent of the PMI, up to at least 36 hours. The RNA quality for specific donated brains could not be predicted based on the PMI. Individual samples with a low PMI could have a poor RIN value, while a sample with a PMI over 36 hours may have a high RIN value. The RIN values for control brain donors, all of whom died suddenly and unexpectedly, were marginally higher than for individuals with clinical brain disorders. Polymerase chain reaction (PCR) analysis of samples confirmed that RIN values were more critical than PMI for determining suitability of tissue for molecular biological studies and samples should be matched by their RIN values rather than PMI. Importantly, PCR analysis established that tissue stored up to 23 years at -80°C yielded high-quality RNA. These results confirm that postmortem human brain tissue collected by brain and tissue banks over decades can serve as high quality material for the study of human disorders.

Effects of Early Alcohol Exposure on Functional Organization and Microstructure of a Visual-Tactile Integrative Circuit.

BACKGROUND: Children with fetal alcohol spectrum disorders (FASD) often have deficits associated with multisensory processing. Because ethanol (EtOH) disrupts activity-dependent neuronal plasticity, a process that is essential for refining connections during cortical development, we hypothesize that early alcohol exposure results in alterations in multisensory cortical networks, which could explain the multisensory processing deficits seen in FASD. Here, we use a gyrencephalic animal model to test the prediction that early alcohol exposure alters the functional connectivity and microstructural features of the rostral posterior parietal cortex (PPr), a visual-tactile integrative area. METHODS: Ferrets were exposed to moderate doses of EtOH during the brain growth spurt period. Functional connectivity and microstructural features were assessed using resting-state functional magnetic resonance imaging and ex vivo diffusion kurtosis imaging (DKI), respectively, when the animals reached juvenile age and adulthood, respectively. RESULTS: While the whole brain volume was smaller in alcohol-treated animals, the relative size of the frontal brain area was larger when compared to control animals. Altered functional connectivity was observed in alcohol-treated animals, where increased connectivity was observed between PPr and the region that provides its major visual inputs (the caudal portion of the parietal cortex), but not with the region that provides its major somatosensory inputs (tertiary somatosensory cortex). DKI revealed reduced microstructural tissue complexity in all investigated sensory areas of alcohol-treated animals. CONCLUSIONS: In this study, we observed alterations in cortical functional connectivity and microstructural integrity in a cortical area involved in multisensory processing in a ferret FASD model. These findings indicate an alteration in cortical networks that may be related to the multisensory processing deficiencies observed in FASD.

Proceedings of the 2016 annual meeting of the Fetal Alcohol Spectrum Disorders Study Group.

The 2016 Fetal Alcohol Spectrum Disorders Study Group (FASDSG) meeting was titled "Rehabilitation in FASD: Potential Interventions and Challenges". During the previous decades, studies with human subjects and animal models have improved much of our understanding of the mechanisms underlying FASD, putting the scientific community in a good position to test hypotheses that can lead to potential therapeutic interventions. During the conference, two keynote speakers addressed potential interventions used in different fields and their applicability to FASD research. The conference also included updates from several government agencies, short presentations by junior and senior investigators that showcased the latest in FASD research, and award presentations. The conference was closed by a talk by Dr. Charles Goodlett, the recipient of the 2016 Henry Rosett award.

The Role of CREB, SRF, and MEF2 in Activity-Dependent Neuronal Plasticity in the Visual Cortex.

The transcription factors CREB (cAMP response element binding factor), SRF (serum response factor), and MEF2 (myocyte enhancer factor 2) play critical roles in the mechanisms underlying neuronal plasticity. However, the role of the activation of these transcription factors in the different components of plasticity in vivo is not well known. In this study, we tested the role of CREB, SRF, and MEF2 in ocular dominance plasticity (ODP), a paradigm of activity-dependent neuronal plasticity in the visual cortex. These three proteins bind to the synaptic activity response element (SARE), an enhancer sequence found upstream of many plasticity-related genes (Kawashima et al., 2009; Rodríguez-Tornos et al., 2013), and can act cooperatively to express Arc, a gene required for ODP (McCurry et al., 2010). We used viral-mediated gene transfer to block the transcription function of CREB, SRF, and MEF2 in the visual cortex, and measured visually evoked potentials in awake male and female mice before and after a 7 d monocular deprivation, which allowed us to examine both the depression component (Dc-ODP) and potentiation component (Pc-ODP) of plasticity independently. We found that CREB, SRF, and MEF2 are all required for ODP, but have differential effects on Dc-ODP and Pc-ODP. CREB is necessary for both Dc-ODP and Pc-ODP, whereas SRF and MEF2 are only needed for Dc-ODP. This finding supports previous reports implicating SRF and MEF2 in long-term depression (required for Dc-ODP), and CREB in long-term potentiation (required for Pc-ODP).SIGNIFICANCE STATEMENT Activity-dependent neuronal plasticity is the cellular basis for learning and memory, and it is crucial for the refinement of neuronal circuits during development. Identifying the mechanisms of activity-dependent neuronal plasticity is crucial to finding therapeutic interventions in the myriad of disorders where it is disrupted, such as Fragile X syndrome, Rett syndrome, epilepsy, major depressive disorder, and autism spectrum disorder. Transcription factors are essential nuclear proteins that trigger the expression of gene programs required for long-term functional and structural plasticity changes. Our results elucidate the specific role of the transcription factors CREB, SRF, and MEF2 in the depression and potentiation components of ODP in vivo, therefore better informing future attempts to find therapeutic targets for diseases where activity-dependent plasticity is disrupted.

Structural and Functional Integrity of the Intraparietal Sulcus in Moderate and Severe Traumatic Brain Injury.

Severe and moderate traumatic brain injury (sTBI) often results in long-term cognitive deficits such as reduced processing speed and attention. The intraparietal sulcus (IPS) is a neocortical structure that plays a crucial role in the deeply interrelated processes of multi-sensory processing and top down attention. Therefore, we hypothesized that disruptions in the functional and structural connections of the IPS may play a role in the development of such deficits. To examine these connections, we used resting state magnetic resonance imaging (rsfMRI and diffusion kurtosis imaging (DKI) in a cohort of 27 patients with sTBI (29.3 ± 8.9 years) and 27 control participants (29.8 ± 10.3 years). Participants were prospectively recruited and received rsfMRI and neuropsychological assessments including the Automated Neuropsychological Assessment Metrics (ANAM) at greater than 6 months post-injury. A subset of participants received a DKI scan. Results suggest that patients with sTBI performed worse than control participants on multiple subtests of the ANAM suggesting reduced cognitive performance. Reduced resting state functional connectivity between the IPS and cortical regions associated with multi-sensory processing and the dorsal attention network was observed in the patients with sTBI. The patients also showed reduced structural integrity of the superior longitudinal fasciculus (SLF), a key white matter tract connecting the IPS to anterior frontal areas, as measured by reduced mean kurtosis (MK) and fractional anisotropy (FA) and increased mean diffusivity (MD). Further, this reduced structural integrity of the SLF was associated with a reduction in overall cognitive performance. These findings suggest that disruptions in the structural and functional connectivity of the IPS may contribute to chronic cognitive deficits experienced by these patients.

Cortical multisensory connectivity is present near birth in humans.

How the newborn brain adapts to its new multisensory environment has been a subject of debate. Although an early theory proposed that the brain acquires multisensory features as a result of postnatal experience, recent studies have demonstrated that the neonatal brain is already capable of processing multisensory information. For multisensory processing to be functional, it is a prerequisite that multisensory convergence among neural connections occur. However, multisensory connectivity has not been examined in human neonates nor are its location(s) or afferent sources understood. We used resting state functional MRI (fMRI) in two independent cohorts of infants to examine the functional connectivity of two cortical areas known to be multisensory in adults: the intraparietal sulcus (IPS) and the superior temporal sulcus (STS). In the neonate, the IPS was found to demonstrate significant functional connectivity with visual association and somatosensory association areas, while the STS showed significant functional connectivity with the visual association areas, primary auditory cortex, and somatosensory association areas. Our findings establish that each of these areas displays functional communication with cortical regions representing various sensory modalities. This demonstrates the presence of cortical areas with converging sensory inputs, representing that the functional architecture needed for multisensory processing is already present within the first weeks of life.

Effects of developmental alcohol and valproic acid exposure on play behavior of ferrets.

Exposure to alcohol and valproic acid (VPA) during pregnancy can lead to fetal alcohol spectrum disorders and fetal valproate syndrome, respectively. Altered social behavior is a hallmark of both these conditions and there is ample evidence showing that developmental exposure to alcohol and VPA affect social behavior in rodents. However, results from rodent models are somewhat difficult to translate to humans owing to the substantial differences in brain development, morphology, and connectivity. Since the cortex folding pattern is closely related to its specialization and that social behavior is strongly influenced by cortical structures, here we studied the effects of developmental alcohol and VPA exposure on the play behavior of the ferret, a gyrencephalic animal known for its playful nature. Animals were injected with alcohol (3.5g/kg, i.p.), VPA (200mg/kg, i.p.) or saline (i.p) every other day during the brain growth spurt period, between postnatal days 10 and 30. The play behavior of pairs of the same experimental group was evaluated 3 weeks later. Both treatments induced significant behavioral differences compared to controls. Alcohol and VPA exposed ferrets played less than saline treated ones, but while animals from the alcohol group displayed a delay in start playing with each other, VPA treated ones spent most of the time close to one another without playing. These findings not only extend previous results on the effects of developmental exposure to alcohol and VPA on social behavior, but make the ferret a great model to study the underlying mechanisms of social interaction.

Astrocytes Assemble Thalamocortical Synapses by Bridging NRX1α and NL1 via Hevin.

Proper establishment of synapses is critical for constructing functional circuits. Interactions between presynaptic neurexins and postsynaptic neuroligins coordinate the formation of synaptic adhesions. An isoform code determines the direct interactions of neurexins and neuroligins across the synapse. However, whether extracellular linker proteins can expand such a code is unknown. Using a combination of in vitro and in vivo approaches, we found that hevin, an astrocyte-secreted synaptogenic protein, assembles glutamatergic synapses by bridging neurexin-1alpha and neuroligin-1B, two isoforms that do not interact with each other. Bridging of neurexin-1alpha and neuroligin-1B via hevin is critical for the formation and plasticity of thalamocortical connections in the developing visual cortex. These results show that astrocytes promote the formation of synapses by modulating neurexin/neuroligin adhesions through hevin secretion. Our findings also provide an important mechanistic insight into how mutations in these genes may lead to circuit dysfunction in diseases such as autism.

Developmental alcohol exposure leads to a persistent change on astrocyte secretome.

Fetal alcohol spectrum disorder is the most common cause of mental disabilities in the western world. It has been quite established that acute alcohol exposure can dramatically affect astrocyte function. Because the effects of early alcohol exposure on cell physiology can persist into adulthood, we tested the hypothesis that ethanol exposure in ferrets during a period equivalent to the last months of human gestation leads to persistent changes in astrocyte secretome in vitro. Animals were treated with ethanol (3.5 g/kg) or saline between postnatal day (P)10-30. At P31, astrocyte cultures were made and cells were submitted to stable isotope labeling by amino acids. Twenty-four hour conditioned media of cells obtained from ethanol- or saline-treated animals (ET-CM or SAL-CM) were collected and analyzed by quantitative mass spectrometry in tandem with liquid chromatography. Here, we show that 65 out of 280 quantifiable proteins displayed significant differences comparing ET-CM to SAL-CM. Among the 59 proteins that were found to be reduced in ET-CM we observed components of the extracellular matrix such as laminin subunits α2, α4, ß1, ß2, and γ1 and the proteoglycans biglycan, heparan sulfate proteoglycan 2, and lumican. Proteins with trophic function such as insulin-like growth factor binding protein 4, pigment epithelium-derived factor, and clusterin as well as proteins involved on modulation of proteolysis such as metalloproteinase inhibitor 1 and plasminogen activator inhibitor-1 were also reduced. In contrast, pro-synaptogeneic proteins like thrombospondin-1, hevin as well as the modulator of extracelular matrix expression, angiotensinogen, were found increased in ET-CM. The analysis of interactome maps through ingenuity pathway analysis demonstrated that the amyloid beta A4 protein precursor, which was found reduced in ET-CM, was previously shown to interact with ten other proteins that exhibited significant changes in the ET-CM. Taken together our results strongly suggest that early exposure to teratogens such as alcohol may lead to an enduring change in astrocyte secretome. Despite efforts in prevention, fetal alcohol spectrum disorders are a major cause of mental disabilities. Here, we show that developmental exposure to alcohol lead to a persistent change in the pattern of proteins secreted (secretome) by astrocytes. This study is also the first mass spectrometry-based assessment of the astrocyte secretome in a gyrencephalic animal. Cover Image for this issue: doi: 10.1111/jnc.13320.