Andrea J. Elberger

Double-labeling of tissue containing the carbocyanine dye DiI for immunocytochemistry

The fluorescent carbocyanine dye DiI can be used for retrograde and anterograde labeling of neuronal pathways. To investigate the possible neurochemical identity of DiI-labeled neuronal cell bodies and terminals, we used a procedure for double-labeling of the same tissue with antisera to specific neuroactive substances. This procedure involves visualizing the immunohistochemical label with an FITC-conjugated secondary antiserum. Both labels can be viewed in the same tissue by fluorescence microscopy, and individual cell bodies and processes double-labeled with DiI and antiserum can be identified by switching between filter sets appropriate for rhodamine (to see the DiI labeling) and for fluorescein (to see the immunhistochemical labeling). The method has been used with primary antisera to excitatory and inhibitory amino acid neurotransmitters, as well as to neuropeptides, and is likely to be useful with antibodies against a wide variety of substances. Several other immunocytochemical methods were found to be incompatible with DiI labeling.

A Novel Closed-Head Model of Mild Traumatic Brain Injury Caused by Primary Overpressure Blast to the Cranium Produces Sustained Emotional Deficits in Mice

Emotional disorders are a common outcome from mild traumatic brain injury (TBI) in humans, but their pathophysiological basis is poorly understood. We have developed a mouse model of closed-head blast injury using an air pressure wave delivered to a small area on one side of the cranium, to create mild TBI. We found that 20-psi blasts in 3-month-old C57BL/6 male mice yielded no obvious behavioral or histological evidence of brain injury, while 25–40 psi blasts produced transient anxiety in an open field arena but little histological evidence of brain damage. By contrast, 50–60 psi blasts resulted in anxiety-like behavior in an open field arena that became more evident with time after blast. In additional behavioral tests conducted 2–8 weeks after blast, 50–60 psi mice also demonstrated increased acoustic startle, perseverance of learned fear, and enhanced contextual fear, as well as depression-like behavior and diminished prepulse inhibition. We found no evident cerebral pathology, but did observe scattered axonal degeneration in brain sections from 50 to 60 psi mice 3–8 weeks after blast. Thus, the TBI caused by single 50–60 psi blasts in mice exhibits the minimal neuronal loss coupled to “diffuse” axonal injury characteristic of human mild TBI. A reduction in the abundance of a subpopulation of excitatory projection neurons in basolateral amygdala enriched in Thy1 was, however, observed. The reported link of this neuronal population to fear suppression suggests their damage by mild TBI may contribute to the heightened anxiety and fearfulness observed after blast in our mice. Our overpressure air blast model of concussion in mice will enable further studies of the mechanisms underlying the diverse emotional deficits seen after mild TBI.

Second trimester prenatal alcohol exposure alters development of rat corpus callosum

Prenatal alcohol exposure produces many developmental defects of the central nervous system (CNS), such as in the corpus callosum (CC). This study was designed to observe the effect of prenatal alcohol exposure during the second trimester equivalent on the development of dendritic arbors of CC projection neurons (CCpn) in rat visual cortex. In addition, the effect of second trimester equivalent prenatal alcohol exposure on brain weight was determined. Pregnant dams received 1.2-6.0 g/kg ethanol (EtOH) during gestational day (G) 11-20. Controls consisted of normal and nutritionally matched pairfed (PF) dams. Pups were sacrificed on the day of birth, G26, G29 and G33. DiI crystals were placed in the midsagittal CC bundle to retrogradely label CCpn. Images of visual cortex were obtained from tissue slices using a confocal laser scanning microscope. The number and length of apical and basilar dendrite branches were determined. The results show that prenatal alcohol exposure restricted to the second trimester equivalent alters the development of the CCpn dendritic arbor and the brain weight in a blood alcohol concentration (BAC)-dependent manner. The alteration in the EtOH CCpn is manifested as an increase in the number and length of CCpn apical and basilar dendrite branches, while brain weight is reduced compared with Controls.

Motor, Visual and Emotional Deficits in Mice after Closed-Head Mild Traumatic Brain Injury Are Alleviated by the Novel CB2 Inverse Agonist SMM-189

We have developed a focal blast model of closed-head mild traumatic brain injury (TBI) in mice. As true for individuals that have experienced mild TBI, mice subjected to 50–60 psi blast show motor, visual and emotional deficits, diffuse axonal injury and microglial activation, but no overt neuron loss. Because microglial activation can worsen brain damage after a concussive event and because microglia can be modulated by their cannabinoid type 2 receptors (CB2), we evaluated the effectiveness of the novel CB2 receptor inverse agonist SMM-189 in altering microglial activation and mitigating deficits after mild TBI. In vitro analysis indicated that SMM-189 converted human microglia from the pro-inflammatory M1 phenotype to the pro-healing M2 phenotype. Studies in mice showed that daily administration of SMM-189 for two weeks beginning shortly after blast greatly reduced the motor, visual, and emotional deficits otherwise evident after 50–60 psi blasts, and prevented brain injury that may contribute to these deficits. Our results suggest that treatment with the CB2 inverse agonist SMM-189 after a mild TBI event can reduce its adverse consequences by beneficially modulating microglial activation. These findings recommend further evaluation of CB2 inverse agonists as a novel therapeutic approach for treating mild TBI.

Corpus callosum and visual cortex of mice with deletion of the NMDA-NR1 receptor. II. Attenuation of prenatal alcohol exposure effects.

Offspring of transgenic mice with deletion of the NMDA-NR1 (NR1) receptor received prenatal alcohol exposure during most of gestation. Before and after birth, offspring were sacrificed in order to examine the morphological consequences of the prenatal exposure. Previously, we reported that the dendritic arborization of corpus callosum projection neurons (CCpn) in visual cortex was abnormal in rats given prenatal alcohol exposure; the effects were dose-dependent [Neurotoxicol. Teratol. 24 (2002) 719-732]. The same parameters were examined in the transgenic mice. Crystals of DiI were placed into the CC of mice at different ages that had been prenatally exposed to alcohol. Controls included untreated transgenic mice, and transgenic mice with the same nutritional and handling stressors. Compared to Controls, prenatal alcohol exposure caused the NR1+/+ mice to expand the dendritic arbor of CCpn in visual cortex. The dendritic arbors had increased branch numbers and length; these increases were dose-dependent. In contrast, the prenatally exposed NR1-/- mice showed normal dendritic arbors with all prenatal alcohol doses. In addition, prenatal alcohol exposure was found to have morbidity and teratogenic effects on offspring. In seven separate indicators of the effects of prenatal alcohol exposure, only one indicator was present but reduced in NR1-/- offspring, indicating that total deletion of the NMDA-NR1 receptor throughout development largely blocks but sometimes attenuates the effects of prenatal alcohol exposure. Similarly, in seven separate indicators of the effects of prenatal alcohol exposure, five indicators were attenuated in NR1+/- compared to NR1+/+ offspring, although affected more than in NR1-/-; this suggests a gene-dose effect. The results indicate that functional NMDA-NR1 receptors are necessary for the neurotoxic and teratogenic effects of prenatal alcohol exposure. This study will aid in understanding how the NMDA receptors play an important role in prenatal alcohol effects on brain development.

A modification of biotinylated dextran amine histochemistry for labeling the developing mammalian brain

Biotinylated dextran amine (BDA) has proven to be an excellent anterograde tracer in adult mammalian brains, having some advantages over other anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biocytin. However, results are inferior when BDA is used in neonatal mammals. To improve the sensitivity and quality of BDA labeling in neonatal mammalian brains, the tetramethylbenzidine-sodium tungstate (TMB-ST) method for horseradish peroxidase (HRP) histochemistry was modified and used in BDA histochemistry. After BDA application to the visual cortex of neonatal rat and cat, contralateral and ipsilateral cortical and subcortical regions were examined for BDA-labeled exons and terminals. The modified BDA histochemistry produced corpus callosum (CC) axons in neonatal rat and cat that were heavily and continuously labeled. The distribution, trajectories, branching and termination of individual CC axons, and even possible axon-axon contracts, were clearly identified in exquisite detail, even at low magnification. The quality of BDA labeling in the ipsilateral lateral geniculate nucleus and superior colliculus was similar to that of the CC axonal labeling. These results indicate that the modified BDA histochemistry provides a very sensitive and reliable approach to revealing the detailed distribution and morphology of projecting axons and terminals in the developing mammalian nervous system.

Selective Labeling of Visual Corpus Callosum Connections with Aspartate in Cat and Rat

Tritiated neurotransmitter candidates were unilaterally injected in visual cortical regions with abundant corpus callosum connections. D-aspartate (Asp) or gamma-aminobutyric acid (GABA) was injected along the area 17/18 border in cat, and the area 17/18a and 17/18b borders in rat. Retrograde Asp label was found contralaterally in supragranular and infragranular laminae in areas 17, 18, 19, PMLS, and PLLS in cat, and in areas 29, 18b, 17, and 18a in rat. No contralateral GABA label was found in cat or rat. Thus, the cat and rat corpus callosum may use Asp or a related substance as a neurotransmitter.

Corpus callosum and visual cortex of mice with deletion of the NMDA-NR1 receptor: I. Accelerated development of callosal projection neurons.

Many pharmacological experiments show that the ionotropic receptor NMDA has both neurotrophic and neuroexcitotoxic effects. The neurotrophic function is manifested in many ways including acceleration of neuronal development, enhancement of neuronal migration, neuroprotection, blockage of apoptosis, prevention of aging and prematurity, as well as effects on synaptic plasticity and synaptogenesis. On the other hand, the neuroexcitotoxic function is manifested in its role in neurological and psychiatric diseases such as epilepsy, Parkinsons disease and schizophrenia. The present study explores the consequences of complete and partial absence of NMDA-NR1 receptors throughout development. Using DiI tracing in vitro, the development of corpus callosum projection neurons in transgenic mice with deletion of the NMDA-NR1 receptor was observed in visual cortex. Compared to littermate controls, the histogenesis and neuronal development of corpus callosum cells of origin was found to be accelerated in NR1-/- mice. That is, the corpus callosum projection neurons in NR1 knockout mice developed earlier and faster than in littermate heterozygous and wild-type mice. However, the corpus callosum projection neurons in NR1 heterozygous mice developed earlier and faster than in littermate wild-type mice. This suggests that NMDA-NR1 receptors are involved in sequencing and/or temporal regulation of neuronal development, and that there is a gene-dose effect. Studies from other laboratories suggest that the observed phenomenon of prematurity or accelerated development is a direct effect of altered expression of genes found in mice with deletion of the NMDA-NR1 receptor.

Heterozygous deletion of NR1 subunit of the NMDA receptor alters ethanol-related behaviors and regional expression of NR2 subunits in the brain.

NMDA receptors have been hypothesized to play a role in various aspects of ethanol-related phenotypes, notably in ethanol withdrawal. However, the role of each of the specific subunits remains unclear. To address this issue, mice that are heterozygous for the NR1 deletion, and thus have a reduction in functional NMDA receptors, were examined for ethanol consumption and acute ethanol withdrawal. Additionally, mice were examined for the level of vocalization following footshock, and behavior in an elevated plus maze, to determine their responses to stress. In these behavioral tests, NR1 heterozygous mice were shown to consume significantly higher levels of ethanol in the two bottle-choice test showing a possible role for this receptor in ethanol consumption. Analysis of acute withdrawal found that the heterozygous mice exhibit lower levels of handling-induced convulsions consistent with a role in ethanol sensitivity or withdrawal. In contrast, no effects on stress-related phenotypes were detected. Levels of NR2A-NR2D subunits of the NMDA receptor in specific brain regions were compared between NR1+/- mice and wild-type controls to assess whether the behavioral responses were specific to the diminution in NR1 expression or whether these changes could be due to secondary changes in expression of other NMDA subunits. Real-time quantitative PCR, Western blot and immunohistochemistry were used to examine expression levels in the hippocampus, neocortex, striatum and cerebellum. For the majority of the subunits, no differences were found between the wild-type and heterozygous mice in any of the brain regions. However, the NR2B subunit exhibited differences in expression of RNA in the hippocampus and protein levels in multiple brain regions, between wild-type and NR1+/- mice. These results show that NR1 plays a role, through mechanisms as yet unknown, in the expression of NR2 subunits in a region and subtype specific manner. This provides evidence of the effects of altered levels of NR1 expression on ethanol withdrawal and consumption, and suggests that concomitant changes in the levels of NR2B may contribute to that effect.

Confirmation of the existence of transitory corpus callosum axons in area 17 of neonatal cat : an anterograde tracing study using biotinylated dextran amine

Corpus callosum (CC) axons in visual cortex were labeled anterogradely by in vivo biotinylated dextran amine (BDA) in neonatal cat at postnatal day (PND) 6, 10 and 15. Labeled CC axons were distributed throughout the visual cortex including medial area 17. The number of CC axons in medial area 17 increased from PND 6 to PND 10, and then decreased from PND 10 to PND 15. At PND 15, few CC axons could be followed into the grey matter in medial area 17. Thus, BDA labels transitory CC axons that extend through all cortical layers in medial area 17, confirming the results revealed by in vitro DiI labeling.