Steven W. Threlkeld

Developmental Disruptions and Behavioral Impairments in Rats Following In Utero RNAi of Dyx1c1

Developmental malformations of cortex have been shown to co-occur with language, learning, and other cognitive deficits in humans. Rodent models have repeatedly shown that animals with such developmental malformations have deficits related to auditory processing and learning. More specifically, freeze-lesion induced microgyria as well as molecular layer ectopias have been found to impair rapid auditory processing ability in rats and mice. In humans, deficits in rapid auditory processing appear to relate to later impairments of language. Recently, genetic variants of four different genes involved in early brain development have been proposed to associate with an elevated incidence of developmental dyslexia in humans. Three of these, DYX1C1, DCDC2, and KIAA0319, have been shown by in utero RNAi to play a role in neuronal migration in developing neocortex. The present study assessed the effects of in utero RNAi of Dyx1c1 on auditory processing and spatial learning in rats. Results indicate that RNAi of Dyx1c1 is associated with cortical heterotopia and is suggestive of an overall processing deficit of complex auditory stimuli in both juvenile and adult periods (p=.051, one-tail). In contrast, adult data alone reveal a significant processing impairment among RNAi treated subjects compared to shams, indicating an inability for RNAi treated subjects to improve detection of complex auditory stimuli over time (p=.022, one-tail). Further, a subset of RNAi treated rats exhibited hippocampal heterotopia centered in CA1 (in addition to cortical malformations). Malformations of hippocampus were associated with robust spatial learning impairment in this sub-group (p<.01, two-tail). In conclusion, in utero RNAi of Dyx1c1 results in heterogeneous malformations that correspond to distinct behavioral impairments in auditory processing, and spatial learning.

Use of a modified prepulse inhibition paradigm to assess complex auditory discrimination in rodents.

Prepulse inhibition (PPI; also termed startle reduction or reflex modification, see Ref. [H.S. Hoffman, J.R. Ison, Reflex modification in the domain of startle: I. Some empirical findings and their implications for how the nervous system processes sensory input, Psychol. Rev. 87 (1980) 175-189]) provides an efficient and accurate method to assess both simple and complex acoustic discrimination in rodents [J.R. Ison, G.R. Hammond, Modification of the startle reflex in the rat by changes in the auditory and visual environments, J. Comp. Physiol. Psychol. 75 (1971) 435-452]. Assessment of acoustic processing using PPI is less time consuming than operant conditioning paradigms, allows for the testing of many subjects simultaneously, and largely eliminates confounds due to motivation and attention [M. Clark, G. Rosen, P. Tallal, R.H. Fitch, Impaired processing of complex auditory stimuli in rats with induced cerebrocortical microgyria, J. Cog. Neurosci. 12 (2000) 828-839]. Moreover, PPI procedures allow for data acquisition from the first day of testing, and can be used on rats as young as P14-15 [J.T. Friedman, A. Peiffer, M. Clark, A. Benasich, R.H. Fitch, Age and experience related improvements in gap detection in the rat, Dev. Brain Res. 152 (2004) 83-91; M. McClure, S. Threlkeld, G. Rosen, R.H. Fitch, Rapid auditory processing and learning deficits in rats with P1 versus P7 neonatal hypoxic-ischemic injury, Behav. Brain Res. 172 (2006) 114-121; S.W. Threlkeld, M.M. McClure, G.D. Rosen, R.H. Fitch, Developmental timeframes for the induction of microgyria and rapid auditory processing deficits in the rat, Brain Res. 1109 (2006) 22-31]. For these and additional reasons, the PPI paradigm has more recently been adapted to the assessment of complex acoustic discrimination (tone sequences and FM sweeps), and applied to the study of normally developing as well as neuropathologically affected rodent populations. The purpose of the current review is to provide a background on the PPI paradigm, and to summarize what has been learned more recently using modified versions of PPI with rodent models.

Reprint of "Early testosterone modulated sex differences in behavioral outcome following neonatal hypoxia ischemia in rats".

Hypoxia ischemia (HI; reduced blood oxygenation and/or flow to the brain) represents one of the most common injuries for both term and preterm/very low birth weight (VLBW) infants. These children experience elevated incidence of cognitive and/or sensory processing disabilities, including language based learning disabilities. Clinical data also indicate more substantial long‐term deficits for HI injured male babies as compared to HI injured females. Previously, we reported significant deficits in rapid auditory processing and spatial learning in male rats with postnatal day 1 (P1), P7, or P10 HI injury. We also showed sex differences in HI injured animals, with more severe deficits in males as compared to females. Given these findings, combined with extant clinical data, the current study sought to assess a putative role for perinatal testosterone in modulating behavioral outcome following early hypoxic‐ischemic injury in rats. Male, female, and testosterone‐propionate (TP) treated females were subjected to P7 HI or sham surgery, and subsequently (P30+) underwent a battery of auditory testing and water maze assessment. Results confirm previous reports of sex differences following HI, and add new findings of significantly worse performance in TP‐treated HI females compared to vehicle treated HI females. Post mortem anatomic analyses showed consistent effects, with significant brain weight decreases seen in HI male and TP‐treated HI females but not female HI or sham groups. Further neuromorphometric analysis of brain structures showed that HI male animals exhibited increased pathology relative to HI females as reflected in ventricular enlargement. Findings suggest that neonatal testosterone may act to enhance the deleterious consequences of early HI brain injury, as measured by both neuropathology and behavior.

Auditory processing and learning/memory following erythropoietin administration in neonatally hypoxic–ischemic injured rats

BACKGROUND Hypoxia-ischemia (HI) is a common injury arising from prematurity/complications at birth and is associated with later language, auditory, and learning impairments. OBJECTIVE To investigate the efficacy of two doses (300 or 1000 U/kg) of Erythropoietin (Epo) in protecting against neuropathological and behavioral impairments associated with HI injury in rats. METHODS HI injury (right carotid artery cauterization and 120 min of 8% O(2)) was induced on postnatal day 7 (P7) and Epo or saline was administered i.p. immediately following the procedure. Auditory processing and learning/memory were assessed throughout development. RESULTS Both doses of Epo provided behavioral protection following HI injury. Rats given 300 or 1000 U/kg of Epo performed significantly better than HI animals on a short duration complex auditory processing procedure, on a spatial Morris water maze assessing spatial learning/reference memory, and a non-spatial water maze assessing associative learning/reference memory. CONCLUSIONS Given Epos extant clinical use (FDA approved for pediatric patients with anemia secondary to prematurity), the current results add to a growing body of literature supporting the use of Epo as a potential protective agent for neurological and behavioral impairments following early HI injury in infants.

Ischemia-reperfusion impairs blood-brain barrier function and alters tight junction protein expression in the ovine fetus

The blood-brain barrier is a restrictive interface between the brain parenchyma and the intravascular compartment. Tight junctions contribute to the integrity of the blood-brain barrier. Hypoxic-ischemic damage to the blood-brain barrier could be an important component of fetal brain injury. We hypothesized that increases in blood-brain barrier permeability after ischemia depend upon the duration of reperfusion and that decreases in tight junction proteins are associated with the ischemia-related impairment in blood-brain barrier function in the fetus. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (K(i)) and tight junction proteins by Western immunoblot in fetal sheep at 127 days of gestation without ischemia, and 4, 24, or 48 h after ischemia. The largest increase in K(i) (P<0.05) was 4 h after ischemia. Occludin and claudin-5 expressions decreased at 4 h, but returned toward control levels 24 and 48 h after ischemia. Zonula occludens-1 and -2 decreased after ischemia. Inverse correlations between K(i) and tight junction proteins suggest that the decreases in tight junction proteins contribute to impaired blood-brain barrier function after ischemia. We conclude that impaired blood-brain barrier function is an important component of hypoxic-ischemic brain injury in the fetus, and that increases in quantitatively measured barrier permeability (K(i)) change as a function of the duration of reperfusion after ischemia. The largest increase in permeability occurs 4 h after ischemia and blood-brain barrier function improves early after injury because the blood-brain barrier is less permeable 24 and 48 than 4 h after ischemia. Changes in the tight junction molecular composition are associated with increases in blood-brain barrier permeability after ischemia.

The effects of erythropoietin on auditory processing following neonatal hypoxic-ischemic injury

Neonatal hypoxia-ischemia (HI) is a common cause of brain damage and subsequent behavioral deficits in premature/term infants. Rapid auditory processing deficits have been suggested to play a role in later language impairments in this population. We have previously shown auditory deficits in rats with neonatal HI injury and now report novel effects of behavioral sparing and neuroprotection following treatment with a low dose of Erythropoietin using this HI injury model.

Persistent spatial working memory deficits in rats following in utero RNAi of Dyx1c1

Disruptions in the development of the neocortex are associated with cognitive deficits in humans and other mammals. Several genes contribute to neocortical development, and research into the behavioral phenotype associated with specific gene manipulations is advancing rapidly. Findings include evidence that variants in the human gene DYX1C1 may be associated with an increased risk of developmental dyslexia. Concurrent research has shown that the rat homolog for this gene modulates critical parameters of early cortical development, including neuronal migration. Moreover, recent studies have shown auditory processing and spatial learning deficits in rats following in utero transfection of an RNA interference (RNAi) vector of the rat homolog Dyx1c1 gene. The current study examined the effects of in utero RNAi of Dyx1c1 on working memory performance in Sprague–Dawley rats. This task was chosen based on the evidence of short‐term memory deficits in dyslexic populations, as well as more recent evidence of an association between memory deficits and DYX1C1 anomalies in humans. Working memory performance was assessed using a novel match‐to‐place radial water maze task that allows the evaluation of memory for a single brief (∼4–10 seconds) swim to a new goal location each day. A 10‐min retention interval was used, followed by a test trial. Histology revealed migrational abnormalities and laminar disruption in Dyx1c1 RNAi‐treated rats. Dyx1c1 RNAi‐treated rats exhibited a subtle, but significant and persistent impairment in working memory as compared to Shams. These results provide further support for the role of Dyx1c1 in neuronal migration and working memory.

Ovine Proinflammatory Cytokines Cross the Murine Blood-Brain Barrier by a Common Saturable Transport Mechanism

Objectives: The cytokines interleukin (IL)-1β and IL-6 are modulators of the neuroimmune axis and have been implicated in neuronal cell death cascades after ischemia or infection. Previous work has shown that some cross-species conservation exists between human and rodent blood-brain barrier (BBB) transport systems. To further assess cross-species conservation of cytokine transport across the BBB, the current studies investigated permeability and inhibition of ovine IL-1β and IL-6 in the mouse. Methods: IL-1β or IL-6 was radioactively labeled with 131I and injected into the jugular vein at time zero. A subset of mice received 1 or 3 µg/mouse of an unlabeled ovine or murine cytokine (IL-1β or IL-6) to assess self- and/or cross-inhibition of transport. Permeability was assessed using multiple-regression analysis. Results: There was a significant linear relationship for both ovine 131I-IL-1β and 131I-IL-6 between brain/serum ratios and exposure time, indicating BBB permeability. Inclusion of 3 µg/mouse unlabeled ovine IL-1β or IL-6 significantly reduced the transport of ovine 131I-IL-1β or 131I-IL-6, respectively, across the BBB. Transport of both ovine 131I-IL-1β and 131I-IL-6 was significantly inhibited by 1 µg/mouse of murine IL-1β or IL-6, respectively. In contrast, 1 µg/mouse of unlabeled ovine IL-1β or IL-6 did not inhibit the transport of murine 131I-IL-1β or 131I-IL-6. Conclusions: Ovine IL-1β and IL-6 cross the mouse BBB by saturable transport. Inhibition of transport by murine homologs indicates that both species use the same transport mechanisms. Conversely, an inability of ovine cytokines to significantly inhibit the transport of murine cytokines indicates that mouse BBB has a lower affinity for ovine than murine cytokines. Knowledge of species-conserved BBB transport mechanisms may facilitate the development of novel animal models of central nervous system pathogenesis.

Detection of silent gaps in white noise following cortical deactivation in rats

Rodent studies using cortical removal techniques, ranging from transient deactivation to surgical ablation of cortex, reveal the importance of auditory cortical integrity in detecting short silent gaps in white noise (2–15 ms). Processing limits for longer gaps under decorticate conditions in rats remain unknown. Determining the temporal threshold for subcortical resolution of gaps in noise could, however, shed light on both normal hierarchical processing of acoustic temporal stimuli, as well as the etiology of processing anomalies following developmental cortical disruption. To address these important issues, we assessed whether intact rats, as well as those with induced developmental cortical disruptions (microgyria) could resolve silent gaps of 20–100 ms in duration when embedded in white noise, during functional deactivation of auditory cortex. Results showed that both intact rats, as well as those with cortical malformations resulting from early focal disruptions of neuronal migration could resolve silent gaps of 100-ms duration under cortical deactivation (KCl). However, only intact rats could reliably detect 75-ms gaps, suggesting possible subcortical anomalies in subjects with early cortical disturbances.

Early acoustic discrimination experience ameliorates auditory processing deficits in male rats with cortical developmental disruption

Auditory temporal processing deficits have been suggested to play a causal role in language learning impairments, and evidence of cortical developmental anomalies (microgyria (MG), ectopia) has been reported for language‐impaired populations. Rodent models have linked these features, by showing deficits in auditory temporal discrimination for rats with neuronal migration anomalies (MG, ectopia). Since evidence from human studies suggests that training with both speech and non‐speech acoustic stimuli may improve language performance in developmentally language‐disabled populations, we were interested in whether/how maturation and early experience might influence auditory processing deficits seen in male rats with induced focal cortical MG.