Protocol for highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence in rodents.

We present a protocol to induce Cre-dependent transgene expression in specific cell types in the rat brain, suppressing a leak expression in off-target cells, by using a flip-excision switch system with a unilateral spacer sequence. We describe steps for construction of transfer plasmids, preparation of adeno-associated viral vectors, intracranial injection, and detection of transgene expression. Our protocol provides a useful strategy for a better understanding of the structure and function of specific cell types in the complex neural circuit. For complete details on the use and execution of this protocol, please refer to Matsushita et al.1.

Highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence.

The flip-excision switch (FLEX) system with an adeno-associated viral (AAV) vector allows expression of transgenes in specific cell populations having Cre recombinase. A significant issue with this system is non-specific expression of transgenes in tissues after vector injection. We show here that Cre-independent recombination events in the AAV genome carrying the FLEX sequence occur mainly during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress the recombination in the viral genome, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our FLEX/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.

Motor skills mediated through cerebellothalamic tracts projecting to the central lateral nucleus.

The cerebellum regulates complex animal behaviors, such as motor control and spatial recognition, through communication with many other brain regions. The major targets of the cerebellar projections are the thalamic regions including the ventroanterior nucleus (VA) and ventrolateral nucleus (VL). Another thalamic target is the central lateral nucleus (CL), which receives the innervations mainly from the dentate nucleus (DN) in the cerebellum. Although previous electrophysiological studies suggest the role of the CL as the relay of cerebellar functions, the kinds of behavioral functions mediated by cerebellothalamic tracts projecting to the CL remain unknown. Here, we used immunotoxin (IT) targeting technology combined with a neuron-specific retrograde labeling technique, and selectively eliminated the cerebellothalamic tracts of mice. We confirmed that the number of neurons in the DN was selectively decreased by the IT treatment. These IT-treated mice showed normal overground locomotion with no ataxic behavior. However, elimination of these neurons impaired motor coordination in the rotarod test and forelimb movement in the reaching test. These mice showed intact acquisition and flexible change of spatial information processing in the place discrimination, Morris water maze, and T-maze tests. Although the tract labeling indicated the existence of axonal collaterals of the DN-CL pathway to the rostral part of the VA/VL complex, excitatory lesion of the rostral VA/VL did not show any significant alterations in motor coordination or forelimb reaching, suggesting no requirement of axonal branches connecting to the VL/VA complex for motor skill function. Taken together, our data highlight that the cerebellothalamic tracts projecting to the CL play a key role in the control of motor skills, including motor coordination and forelimb reaching, but not spatial recognition and its flexibility.

Enhancement of the transduction efficiency of a lentiviral vector for neuron-specific retrograde gene delivery through the point mutation of fusion glycoprotein type E.

BACKGROUND: Pseudotyping of a lentiviral vector with fusion glycoproteins composed of rabies virus glycoprotein (RVG) and vesicular stomatitis virus glycoprotein (VSVG) segments achieves high gene transfer efficiency through retrograde transport in the nervous system. In our previous study, we determined the junction of RVG/VSVG segments of glycoproteins that enhances the transduction efficiency of the neuron-specific retrograde gene transfer (NeuRet) vector (termed fusion glycoprotein type E or FuG-E). NEW METHOD: We aimed to optimize the amino acid residue at position 440 in the membrane-proximal region of FuG-E to improve the efficiency of retrograde gene transfer in the brain. RESULTS: We constructed variants of FuG-E with 18 kinds of single amino acid substitutions at residue 440 to generate lentiviral vectors pseudotyped with these variants, and tested in vivo gene transfer of the vectors in the mouse brain. The FuG-E (P440E) variant, in which proline was substituted by glutamate at residue 440 in FuG-E, showed the greatest retrograde gene transfer efficiency in the brain, bearing the property of the NeuRet vector. The FuG-E (P440E) pseudotype also displayed efficient retrograde gene transfer in the common marmoset brain. COMPARISON WITH EXISTING METHODS: The NeuRet vector with the FuG-E (P440E) variant demonstrated higher retrograde gene transfer efficiency into different neural pathways compared with the parental FuG-E vector. CONCLUSIONS: The FuG-E (P440E) pseudotype provides a powerful tool to investigate neural circuit mechanisms underlying various brain functions and for gene therapy trials of neurological and neurodegenerative diseases.

Action Selection and Flexible Switching Controlled by the Intralaminar Thalamic Neurons.

Learning processes contributing to appropriate selection and flexible switching of behaviors are mediated through the dorsal striatum, a key structure of the basal ganglia circuit. The major inputs to striatal subdivisions are provided from the intralaminar thalamic nuclei, including the central lateral nucleus (CL) and parafascicular nucleus (PF). Thalamostriatal neurons in the PF modulate the acquisition and performance of stimulus-response learning. Here, we address the roles of the CL thalamostriatal neurons in learning processes by using a selective neural pathway targeting technique. We show that the CL neurons are essential for the performance of stimulus-response learning and for behavioral flexibility, including reversal and attentional set-shifting of learned responses. In addition, chemogenetic suppression of neural activity supports the requirements of these neurons for behavioral flexibility. Our results suggest that the main contribution of the CL thalamostriatal neurons is functional control of the basal ganglia circuit linked to the prefrontal cortex.