Microendoscopic calcium imaging of the primary visual cortex of behaving macaques.

In vivo calcium imaging with genetically encoded indicators has recently been applied to macaque brains to monitor neural activities from a large population of cells simultaneously. Microendoscopic calcium imaging combined with implantable gradient index lenses captures neural activities from deep brain areas with a compact and convenient setup; however, this has been limited to rodents and marmosets. Here, we developed miniature fluorescent microscopy to image neural activities from the primary visual cortex of behaving macaques. We found tens of clear fluorescent signals from three of the six brain hemispheres. A subset of these neurons showed clear retinotopy and orientation tuning. Moreover, we successfully decoded the stimulus orientation and tracked the cells across days. These results indicate that microendoscopic calcium imaging is feasible and reasonable for investigating neural circuits in the macaque brain by monitoring fluorescent signals from a large number of neurons.

Evaluation of the expression pattern of rAAV2/1, 2/5, 2/7, 2/8, and 2/9 serotypes with different promoters in the mouse visual cortex.

This study compared the expression pattern, laminar distribution, and cell specificity of several rAAV serotypes (2/1, 2/5, 2/7, 2/8, and 2/9) injected in the primary visual cortex (V1) of adult C57Bl/6J mice. In order to obtain specific expression in certain neuron subtypes, different promoter sequences were evaluated for excitatory cell specificity: a universal cytomegalovirus (CMV) promoter, and two versions of the excitatory neuron-specific Ca(2+) /calmodulin-dependent kinase subunit α (CaMKIIα) promoter, CaMKIIα 0.4 and CaMKIIα 1.3. The spatial distribution as well as the cell type specificity was immunohistochemically verified. Depending on the rAAV serotype used, the transduced volume expressing reporter protein differed substantially (rAAV2/5 ≫ 2/7 ≈ 2/9 ≈ 2/8 ≫ 2/1). Excitatory neuron-specific targeting was promoter-dependent, with a surprising difference between the 1.3 kb and 0.4 kb CaMKIIα promoters. While CaMKIIα 1.3 and CMV carrying vectors were comparable, with 78% of the transduced neurons being excitatory for CMV and 82% for CaMKIIα 1.3, the shorter CaMKIIα 0.4 version resulted in 95% excitatory specificity. This study therefore puts forward the CaMKIIα 0.4 promoter as the best choice to target excitatory neurons with rAAVs. Together, these results can be used as an aid to select the most optimal vector system to deliver transgenes into specific rodent neocortical circuits, allowing further elucidation of their functions.

Multisynaptic inputs from the medial temporal lobe to V4 in macaques.

Retrograde transsynaptic transport of rabies virus was employed to undertake the top-down projections from the medial temporal lobe (MTL) to visual area V4 of the occipitotemporal visual pathway in Japanese monkeys (Macaca fuscata). On day 3 after rabies injections into V4, neuronal labeling was observed prominently in the temporal lobe areas that have direct connections with V4, including area TF of the parahippocampal cortex. Furthermore, conspicuous neuron labeling appeared disynaptically in area TH of the parahippocampal cortex, and areas 35 and 36 of the perirhinal cortex. The labeled neurons were located predominantly in deep layers. On day 4 after the rabies injections, labeled neurons were found in the hippocampal formation, along with massive labeling in the parahippocampal and perirhinal cortices. In the hippocampal formation, the densest neuron labeling was seen in layer 5 of the entorhinal cortex, and a small but certain number of neurons were labeled in other regions, such as the subicular complex and CA1 and CA3 of the hippocampus proper. The present results indicate that V4 receives major input from the hippocampus proper via the entorhinal cortex, as well as "short-cut" pathways that bypass the entorhinal cortex. These multisynaptic pathways may define an anatomical basis for hippocampal-cortical interactions involving lower visual areas. The multisynaptic input from the MTL to V4 is likely to provide mnemonic information about object recognition that is accomplished through the occipitotemporal pathway.

Cortical vision loss as a prominent feature of H1N1 encephalopathy.

A 20-year-old woman infected with the 2009 H1N1 strain of influenza A developed bilateral visual loss. Brain MRI showed restricted diffusion of the parietal and occipital lobes, and her spinal fluid did not contain inflammatory cells. This report describes an unusual case of H1N1 influenza A virus infection primarily affecting the posterior visual pathways.

Parallel feedback pathways in visual cortex of cats revealed through a modified rabies virus.

The visual cortex of cats is highly evolved. Analogously to the brains of primates, large numbers of visual areas are arranged hierarchically and can be parsed into separate dorsal and ventral streams for object recognition and visuospatial representation. Within early primate visual areas, V1 and V2, and to a lesser extent V3, the two streams are relatively segregated and relayed in parallel to higher order cortex, although there is some evidence suggesting an alignment of V2 and V3 to one stream over the other. For cats, there is no evidence of anatomical segregation in areas 18 and 19, the analogs to V2 and V3. However, previous work was only qualitative in nature. Here we re-examined the feedback connectivity patterns of areas 18/19 in quantitative detail. To accomplish this, we used a genetically modified rabies virus that acts as a retrograde tracer and fills neurons with fluorescent protein. After injections into area 19, many more neurons were labeled in putative ventral stream area 21a than in putative dorsal stream region posterolateral suprasylvian complex of areas (PLS), and the dendrites of neurons in 21a were significantly more complex. Conversely, area 18 injections labeled more neurons in PLS, and these were more complex than neurons in 21a. We infer from our results that area 19 in cat is more aligned to the ventral stream and area 18 to the dorsal stream. Based on the success of our approach, we suggest that this method could be applied to resolve similar issues related to primate V3.

Targeting single neuronal networks for gene expression and cell labeling in vivo.

To understand fine-scale structure and function of single mammalian neuronal networks, we developed and validated a strategy to genetically target and trace monosynaptic inputs to a single neuron in vitro and in vivo. The strategy independently targets a neuron and its presynaptic network for specific gene expression and fine-scale labeling, using single-cell electroporation of DNA to target infection and monosynaptic retrograde spread of a genetically modifiable rabies virus. The technique is highly reliable, with transsynaptic labeling occurring in every electroporated neuron infected by the virus. Targeting single neocortical neuronal networks in vivo, we found clusters of both spiny and aspiny neurons surrounding the electroporated neuron in each case, in addition to intricately labeled distal cortical and subcortical inputs. This technique, broadly applicable for probing and manipulating single neuronal networks with single-cell resolution in vivo, may help shed new light on fundamental mechanisms underlying circuit development and information processing by neuronal networks throughout the brain.

Resting cerebral blood flow: a potential biomarker of the effects of HIV in the brain.

OBJECTIVE: HIV enters the brain soon after infection causing neuronal damage and microglial/astrocyte dysfunction leading to neuropsychological impairment. We examined the impact of HIV on resting cerebral blood flow (rCBF) within the lenticular nuclei (LN) and visual cortex (VC). METHODS: This cross-sectional study used arterial spin labeling MRI (ASL-MRI) to measure rCBF within 33 HIV+ and 26 HIV- subjects. Nonparametric Wilcoxon rank sum test assessed rCBF differences due to HIV serostatus. Classification and regression tree (CART) analysis determined optimal rCBF cutoffs for differentiating HIV serostatus. The effects of neuropsychological impairment and infection duration on rCBF were evaluated. RESULTS: rCBF within the LN and VC were significantly reduced for HIV+ compared to HIV- subjects. A 2-tiered CART approach using either LN rCBF < or =50.09 mL/100 mL/min or LN rCBF >50.09 mL/100 mL/min but VC rCBF < or =37.05 mL/100 mL/min yielded an 88% (29/33) sensitivity and an 88% (23/26) specificity for differentiating by HIV serostatus. HIV+ subjects, including neuropsychologically unimpaired, had reduced rCBF within the LN (p = 0.02) and VC (p = 0.001) compared to HIV- controls. A temporal progression of brain involvement occurred with LN rCBF significantly reduced for both acute/early (<1 year of seroconversion) and chronic HIV-infected subjects, whereas rCBF in the VC was diminished for only chronic HIV-infected subjects. CONCLUSION: Resting cerebral blood flow (rCBF) using arterial spin labeling MRI has the potential to be a noninvasive neuroimaging biomarker for assessing HIV in the brain. rCBF reductions that occur soon after seroconversion possibly reflect neuronal or vascular injury among HIV+ individuals not yet expressing neuropsychological impairment.

Rapid, long-term labeling of cells in the developing and adult rodent visual cortex using double-stranded adeno-associated viral vectors.

Chronic in vivo imaging studies of the brain require a labeling method that is fast, long-lasting, efficient, nontoxic, and cell-type specific. Over the last decade, adeno-associated virus (AAV) has been used to stably express fluorescent proteins in neurons in vivo. However, AAV's main limitation for many studies (such as those of neuronal development) is the necessity of second-strand DNA synthesis, which delays peak transgene expression. The development of double-stranded AAV (dsAAV) vectors has overcome this limitation, allowing rapid transgene expression. Here, we have injected different serotypes (1, 2, 6, 7, 8, and 9) of a dsAAV vector carrying the green fluorescent protein (GFP) gene into the developing and adult mouse visual cortex and characterized its expression. We observed labeling of both neurons and astrocytes with serotype-specific tropism. dsAAV-GFP labeling showed high levels of neuronal GFP expression as early as 2 days postinjection and as long as a month, surpassing conventional AAV's onset of expression and matching its longevity. Neurons labeled with dsAAV-GFP appeared structurally and electrophysiologically identical to nonlabeled neurons, suggesting that dsAAV-GFP is neither cytotoxic nor alters normal neuronal function. We also demonstrated that dsAAV-labeled cells can be imaged with subcellular resolution in vivo over multiple days. We conclude that dsAAV is an excellent vector for rapid labeling and long-term in vivo imaging studies of astrocytes and neurons on the single cell level within the developing and adult visual cortex.

Intravitreal gene therapy reduces lysosomal storage in specific areas of the CNS in mucopolysaccharidosis VII mice.

The mucopolysaccharidoses (MPSs) are lysosomal storage diseases resulting from impaired catabolism of sulfated glycosaminoglycans. MPS VII mice lack lysosomal beta-glucuronidase (GUSB) activity, leading to the accumulation of partially degraded chondroitin, dermatan, and heparan sulfates in most tissues. Consequently, these mice develop most of the symptoms exhibited by human MPS VII patients, including progressive visual and cognitive deficits. To investigate the effects of reducing lysosomal storage in nervous tissues, we injected recombinant adeno-associated virus encoding GUSB directly into the vitreous humor of young adult mice. Interestingly, GUSB activity was subsequently detected in the brains of the recipients. At 8-12 weeks after treatment, increased GUSB activity and reduced lysosomal distension were found in regions of the thalamus and tectum that received inputs from the injected eye. Lysosomal storage was also reduced in adjacent nonvisual regions, including the hippocampus, as well as in the visual cortex. The findings suggest that both diffusion and trans-synaptic transfer contribute to the dissemination of enzyme activity within the CNS. Intravitreal injection may thus provide a means of delivering certain therapeutic gene products to specific areas within the CNS.

Herpes simplex virus-mediated gene delivery to the rodent visual system.

PURPOSE: To determine the types of cells in the visual system of the mouse and rat that can express a transgene delivered by an attenuated replication competent Herpes simplex virus-1 (HSV-1) vector. METHODS: C57/BL6 x BALB/C mice and Albino rats were treated with 1 x 10(7) pfu of the HSV-1 ribonucleotide reductase mutant (hrR3) expressing the Escherichia coli lacZ gene. The hrR3 virus was delivered by topical application to the cornea, intravitreal (IV) injection, intracameral injection (IC), or stereotactic injection into the visual cortex (VC). At specified times postinfection, animals were killed and tissues were removed, fixed, sectioned, and stained with X-gal or hematoxylin and eosin for histochemical and histopathologic examination. RESULTS: Topical delivery after corneal scarification in both mouse and rat resulted in lacZ expression in 25% of the corneal epithelial cells and 25% of the retinal pigment epithelium (RPE) cells. Topical application without concurrent scarification also resulted in transgene delivery to 20% of the RPE cells of the rat but not the mouse. IV injection resulted in expression primarily in RPE cells, with up to 100% of the cells expressing lacZ in the mouse and rat. Other cells expressing the transgene included ciliary body (CB) and optic nerve cells. Up to 25% of the retinal ganglion cells in the rat expressed lacZ, but only rarely in mice. IC delivery in rats resulted in expression in trabecular meshwork, CB cells, RPE, and iris epithelium. Injection into area 17 of the rat VC resulted in efficient labeling of the VC neurons and neurons in the lateral geniculate nucleus without any evident pathology or inflammation. Neither inflammation nor disease pathology was observed in either the mouse or rat after any route of delivery. CONCLUSIONS: It was demonstrated that the hrR3 HSV-1 virus can deliver a functioning gene to several cell types in the eye and neurons in the VC and that the virus can move via retrograde transport to nuclei that project to the VC.

PCR detection of host and HIV-1 sequences from archival brain tissue.

Mutations in CCR5 and CCR2b have been recently shown to affect disease progression towards AIDS. A role for these host genotypes in AIDS dementia complex (ADC) has also been postulated but remains unclear. Additionally, brain-derived envelope sequences from HIV-1 have been associated with ADC but their specific contribution to pathogenesis remains uncertain. This study demonstrates the successful use of PCR techniques to isolate host CCR5 and CCR2b, and HIV-1 V3 sequences from paraffin embedded tissues from patients with and without ADC. PCR amplification from archival tissue offers a novel approach for studying the interactions between potential neuroprotective elements in the host and virulence determinants in HIV that may contribute to differences in susceptibility to ADC.