1 Department of PediatricsEffects of early alcohol exposure on visual cortex development and plasticity Alexandre E. Medina, PhD Department of Pediatrics

2 The retino-geniculo- pathwayRetino-geniculo-cortical pathway in humans Retino-geniculo-cortical pathway in rodents

3 What the visual cortex can tell us about FASD?The effects of early alcohol exposure on visual processing (responsiveness, acuity, neuronal orientation tuning). The effects of early alcohol exposure on the functional architecture of the visual cortex (orientation and ocular dominance columns) Neuronal plasticity: The ocular dominance plasticity paradigm

4 Most of the connectivity of the visual system is established during the second half of gestation and first weeks after birth (in humans). Our lab uses a model of “third trimester” alcohol exposure

5 Can we reverse alcohol effects?PART – I: Effects of developmental alcohol exposure on visual cortex properties. PART – II: What the visual cortex tell us about the effects of developmental alcohol exposure on neuronal plasticity? Can we reverse alcohol effects? PART – III: Using viral mediated gene transfer for restoring neuronal plasticity in a ferret model FASD

6 PART – I: Effects of developmental alcohol exposure on visual cortex properties.

8 Orientation selectivity columns and Neuronal orientation tuningMedina et al J. Neurophysiol ; Krahe et al PLoS ONE R L M C LS Alcohol exposure during the equivalent of late gestation in humans does disrupt the functional architecture of the visual cortex and disrupts individual orientation tuning in the ferret.

10 Future directions / Interesting developmentsDoes early alcohol exposure affects retinotopy? From Kalatsky & Stryker (2003), Neuron Jan Does early alcohol exposure affects orientation selectivity in humans ? 5 weeks 7 weeks From Braddick , Wattan-Beel and Atkinson (1986), Nature Newborn

11 PART – II: What the visual cortex tell us about the effects of developmental alcohol exposure on neuronal plasticity?

13 Neuronal plasticity: Processes of Gain and Loss.

14 OD Plasticity is composed by different types of “plasticity”The ocular dominance plasticity paradigm : a model of neuronal plasticity in the cortex Right eye deprived OD Plasticity is composed by different types of “plasticity” Physiological changes – depression or potentiation of synapses Molecular changes – Changes in receptors, number of synapses, changes in gene and protein expression. Morphological changes – Changes in spines, dendritic arborization, axonal terminals and buttons Behavioral – Lost vision

15 Using the Visual Cortex as a model to study the effects of early alcohol exposure on cortical neuronal plasticity The ocular dominance plasticity paradigm Right eye deprived R L M C LS Most of what we know about sensory plasticity came from studies done in visual cortex. In our lab we use the visual cortex as a model to study the effects of early alcohol exposure on cortical neuronal plasticity. The visual cortex, like other sensory systems, presents a marked modular organization which is evident when we look at ocular dominance columns. In Humans, Monkeys, cats and ferrets eye-specific afferents carry visual information from the retina to the lateral geniculate nucleus. They then proceed to layer 4 of the primary visual cortex in alternating ocular dominance bands that are anatomically and physiological dominated by a specific eye. This segregation is most evident in layer IV, however it is also noted in the other layers. Here we see an cartoon of the left hemisphere of an animal. Afferents from the left eye are showed in red and afferents from the right eye are showed in blue. In the 60s David Hubel and Torsten Wiesel showed that during a critical period of development, experience can alter the anatomy and function of the visual cortex. They showed that after a few days of monocular deprivation by lid suture, there is a shift in the ocular dominance profile towards the experienced eye. This shift reflects the shrinkage of connections relaying information from the deprived eye and the expansion of connections relaying information from the experienced eye. This model have been largely studied and share similar mechanisms with other types of plasticity such as learning and memory, LTP and LTD. In our lab we use two methods to assess the ocular dominance columns: Optical imaging of intrinsic signals and single unit recordings. . Optical imaging is based on the fact that activity causes changes in in blood flow and oxygen consumption which lead to changes in light refraction properties. Grossly speaking the color of brain tissue changes after sensory stimulation. A camera take images from cortical areas before and after a stimulus allowing a computer to build a map showing the more active areas. In our preparation an anesthetized ferret is placed in a stereotaxic frame and the visual cortex is exposed. Using a monitor, we present different moving gratings to the left and the right eye. With this method we are able to create a map showing which regions become more active after left or right eye stimulation. Here you are seeing a dorsal view of a ferret visual cortex. Here is rostral, caudal, lateral and medial. Dark regions correspond to areas that respond better to the right eye while white regions correspond to areas that respond better to the left eye. The ocular dominance columns in the ferret are visible in the caudal portion of the occipital cortex. So you can see this patchy distribution here, black , white , black white. So here we see an optical imaging of a ferret after 3 days of monocular deprivation of the Right eye). You can note that the caudal portion of the occipital cortex is completely dominated by the left eye and ocular dominance columns no longer can be seen. Another way to look at ocular dominance is using single-unit recordings. In this approach we present a moving bar of light in a tangent screen. Every time the bar of light crosses the receptive field of a single neuron action potentials are fired and the spikes collected by an electrode placed in the left visual cortex. Here we will show a video made by the nobel prizes David Hubel and Torsten Wiesel showing an example of this method. So presenting a bar of light for each eye separately, you can determine how strongly each eye is connected to a particular neuron in the visual cortex and you can create a ocular dominance profile. The histogram here show the OD profile of a normal ferret. Imagine that you have pass an electrode diagonally through several columns and many layers. At the end of an experiment you will have recorded ocular dominance from many neurons. An index of 0 means that a neuron just responds to the RE whiles an index of 1 means that a neuron responds just to the left eye. Intermediate scores represent binocular cells. As you can note in a normal ferret there is a bias toward the contralateral eye. In single-unit recordings, monocular deprivation results in a pronounced shift towards the experienced eye. Note the lack of cells responding exclusively to the right eye.

16 Early alcohol exposure leads to a long lasting impairment of ODP(Medina, Krahe, Coppola and Ramoa, 2003 Journal of Neuroscience) Ethanol 25% in saline I.P. or Saline as a control Dosage: ml/g (3.5 g/Kg) Goal: reach Blood Alcohol Level (BAL) between mg/dl Frequency: every other day Saline + MD 3 days No MD The period of treatment matches the third trimester of human gestation. In order After the treatment some animals were tested at the period of the peak of plasticity and others after the peak of plasticity. The reason of the first group is to verify if alcohol can block plasticity even when the visual cortex is more plastic. And the reason for the second group is to verify if the effects of alcohol exposure can be reversed with time. Ethanol + MD 3 days

17 How neuronal plasticity is affected in FASD?

18 Phosphodiesterase Inhibitors are good candidates for neuronal plasticity enhancement

19 The impairments caused by alcohol on ODP can be reversed if you treat animals with Vinpocetine a PDE type 1 inhibitor . (Medina, Krahe and Ramoa Journal of Neuroscience)

20 Vinpocetine improves learning in a rat model of FASD.(Filgueiras, Krahe and Medina, 2010, Neurosci letters)

21 CONCLUSION: PDE1 inhibition by Vinpocetine was able to reverse the effects of early alcohol exposure on ocular dominance plasticity, restore the establishment of orientation selectivity columns and improve learning in animal models of FASD (ferrets, rats and mice*).

22 Future directions Can inhibition of PDE1 by Caffeine Improve neuronal plasticity in FASD? 0.4 mg/Kg mg /l mM 2.5 mg/Kg - 2 mg/l – 0.01 mM 20 mg/Kg plus daily supplemental doses of 5 mg/Kg : 20-30% of PDE1 inhibition?

23 PART – III: Using viral mediated gene transfer to restore neuronal plasticity in a ferret model FASD

24 ADDITIONAL READING Restoration of neuronal plasticity by genetic modification Overexpression of serum response factor restores ocular dominance plasticity in a model of fetal alcohol spectrum disorders. Paul, et al. (2010) Journal of Neuroscience

25 Overexpression of serum response factor in astrocytes improves neuronal plasticity in a model of early alcohol exposure. Paul and Medina (2012) Neuroscience

26 Overexpression of SRF in astrocytes improve neuronal plasticity in FASD.What molecules astrocytes are secreting?.

27 THANK YOU!!!! Medina Lab Crystal Lantz Acknowledgments: WELCOME!!Dr. Alexandre Medina Weili Wang Arco Paul Crystal Lantz Acknowledgments: WELCOME!! Dr Pablo Trindade, Post Doc Nisha Pulimood, Graduate student Dr. Rashmin Hingrajia, Fellow Dr Thomas Krahe – VCU Dr Ray Colello - VCU Dr Claudio Filgueiras – UERJ Dr Mriganka Sur – MIT Dr Daniela Tropea – MIT Dr Benjamin Philpot – UNC, Chapel Hill Dr Eric Kandel – Columbia University Fernanda Pohl-Guimaraes – U. of Florida Supported by:

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30 Alcohol + Vinpocetine Saline Alcohol

31 Avaiable at hemifoundation.orgHow plastic is the brain? From Hemispherectomy: How Much Brain Do We Really Need? : Discovery Magazine 1996 Avaiable at hemifoundation.org