Connected neurons in multiple neocortical areas, comprising parallel circuits, encode essential information for visual shape learning.

Neocortical areas comprised of multiple neuronal circuits which are encoded with innumerable advanced cognitive tasks. Studies focused on neuronal network and synaptic plasticity has hypothesized that every specific neuron and the circuit process the explicit essential information for the specific tasks. However, the structure of these circuits and the involved critical neurons remain to be elucidated. Considering our previous studies, showing the specificity of rat postrhinal cortex comprising specific neuronal circuit for encoding both the learning and recall of shape discrimination through a fast neurotransmitter release from the transduced neurons, here we have demonstrated that postsynaptic neurons in two distinct areas, perirhinal cortex and the ventral temporal association areas are required for the specific visual shape discriminations learning. The constitutively active PKC was delivered into neuronal cells in postrhinal cortex, and the animals were allowed to learn the new shape discriminations, and then the silencing siRNA was delivered into postsynaptic neurons in either perirhinal cortex or ventral temporal association areas, using a novel technology for gene transfer into connected neurons. We observed that expression of the siRNA caused the deficits in visual performance, via blocking the activity in the neurons, as displayed by activity-dependent gene imaging, and also subsequently obstructed the activation of specific signaling pathways required for further learning, and dendritic protein synthesis and CREB. Thus, ratifying the conclusion that the two parallel circuits are both required for the visual shape discrimination learning.

Efficient gene transfers into neocortical neurons connected by NMDA NR1-containing synapses.

BACKGROUND: Within a circuit, specific neurons and synapses are hypothesized to have essential roles in circuit physiology and learning, and dysfunction in these neurons and synapses causes specific disorders. These critical neurons and synapses are embedded in complex circuits containing many neuron and synapse types. NEW METHOD: We established technology that can deliver different genes into pre- and post-synaptic neurons connected by a specific synapse type. The first, presynaptic gene transfer employs standard gene transfer technology to express a synthetic peptide neurotransmitter which has three domains, a dense core vesicle sorting domain for processing the protein as a peptide neurotransmitter, a receptor-binding domain, here a small peptide that binds to NMDA NR1 subunits, and the His tag. Upon release, this peptide neurotransmitter binds to its cognate receptor on postsynaptic neurons. Gene transfer selectively into these postsynaptic neurons employs antibody-mediated, targeted gene transfer and anti-His tag antibodies, which recognize the His tag domain in the synthetic peptide neurotransmitter. RESULTS: For the model system, we studied the connection from projection neurons in postrhinal cortex to specific neurons in perirhinal cortex. In our initial report, gene transfer to connected neurons was 20+1% specific. Here, we optimized the technology; we improved the transfection for packaging by using a modern using a modern lipid, Lipofectamine 3000, and used a modern confocal microscope to collect data. We now report 80+2% specific gene transfer to connected neurons. COMPARISON WITH EXISTING METHODS: There is no existing method with this capability. CONCLUSIONS: This technology may enable studies on the roles of specific neurons and synapses in circuit physiology and learning, and support gene therapy treatments for specific disorders.

Delivery of different genes into pre- and post-synaptic neocortical interneurons connected by GABAergic synapses.

Local neocortical circuits play critical roles in information processing, including synaptic plasticity, circuit physiology, and learning, and GABAergic inhibitory interneurons have key roles in these circuits. Moreover, specific neurological disorders, including schizophrenia and autism, are associated with deficits in GABAergic transmission in these circuits. GABAergic synapses represent a small fraction of neocortical synapses, and are embedded in complex local circuits that contain many neuron and synapse types. Thus, it is challenging to study the physiological roles of GABAergic inhibitory interneurons and their synapses, and to develop treatments for the specific disorders caused by dysfunction at these GABAergic synapses. To these ends, we report a novel technology that can deliver different genes into pre- and post-synaptic neocortical interneurons connected by a GABAergic synapse: First, standard gene transfer into the presynaptic neurons delivers a synthetic peptide neurotransmitter, containing three domains, a dense core vesicle sorting domain, a GABAA receptor-binding domain, a single-chain variable fragment anti-GABAA ß2 or ß3, and the His tag. Second, upon release, this synthetic peptide neurotransmitter binds to GABAA receptors on the postsynaptic neurons. Third, as the synthetic peptide neurotransmitter contains the His tag, antibody-mediated, targeted gene transfer using anti-His tag antibodies is selective for these neurons. We established this technology by expressing the synthetic peptide neurotransmitter in GABAergic neurons in the middle layers of postrhinal cortex, and the delivering the postsynaptic vector into connected GABAergic neurons in the upper neocortical layers. Targeted gene transfer was 61% specific for the connected neurons, but untargeted gene transfer was only 21% specific for these neurons. This technology may support studies on the roles of GABAergic inhibitory interneurons in circuit physiology and learning, and support gene therapy treatments for specific disorders associated with deficits at GABAergic synapses.

Separate Gene Transfers into Pre- and Postsynaptic Neocortical Neurons Connected by mGluR5-Containing Synapses.

mGluR5-containing synapses have essential roles in synaptic plasticity, circuit physiology, and learning, and dysfunction at these synapses is implicated in specific neurological disorders. As mGluR5-containing synapses are embedded in large and complex distributed circuits containing many neuron and synapse types, it is challenging to elucidate the roles of these synapses and to develop treatments for the associated disorders. Thus, it would be advantageous to deliver different genes into pre- and postsynaptic neurons connected by a mGluR5-containing synapse. Here, we develop this capability: The first gene transfer, into the presynaptic neurons, uses standard techniques to deliver a vector that expresses a synthetic peptide neurotransmitter. This peptide neurotransmitter has three domains: a dense core vesicle sorting domain, a mGluR5-binding domain composed of a single-chain variable fragment anti-mGluR5, and the His tag. Upon release, this peptide neurotransmitter binds to mGluR5, predominately located on the postsynaptic neurons. Selective gene transfer into these neurons uses antibody-mediated, targeted gene transfer and anti-His tag antibodies, as the synthetic peptide neurotransmitter contains the His tag. For the model system, we studied the connection between neurons in two neocortical areas: postrhinal and perirhinal cortices. Targeted gene transfer was over 80% specific for mGluR5-containing synapses, but untargeted gene transfer was only ~ 15% specific for these synapses. This technology may enable studies on the roles of mGluR5-containing neurons and synapses in circuit physiology and learning and support gene therapy treatments for specific disorders that involve dysfunction at these synapses.

An identified ensemble within a neocortical circuit encodes essential information for genetically-enhanced visual shape learning.

Advanced cognitive tasks are encoded in distributed neocortical circuits that span multiple forebrain areas. Nonetheless, synaptic plasticity and neural network theories hypothesize that essential information for performing these tasks is encoded in specific ensembles within these circuits. Relatively simpler subcortical areas contain specific ensembles that encode learning, suggesting that neocortical circuits contain such ensembles. Previously, using localized gene transfer of a constitutively active protein kinase C (PKC), we established that a genetically-modified circuit in rat postrhinal cortex, part of the hippocampal formation, can encode some essential information for performing specific visual shape discriminations. However, these studies did not identify any specific neurons that encode learning; the entire circuit might be required. Here, we show that both learning and recall require fast neurotransmitter release from an identified ensemble within this circuit, the transduced neurons; we blocked fast release from these neurons by coexpressing a Synaptotagmin I siRNA with the constitutively active PKC. During learning or recall, specific signaling pathways required for learning are activated in this ensemble; during learning, calcium/calmodulin-dependent protein kinase II, MAP kinase, and CREB are activated; and, during recall, dendritic protein synthesis and CREB are activated. Using activity-dependent gene imaging, we showed that during learning, activity in this ensemble is required to recruit and activate the circuit. Further, after learning, during image presentation, blocking activity in this ensemble reduces accuracy, even though most of the rest of the circuit is activated. Thus, an identified ensemble within a neocortical circuit encodes essential information for performing an advanced cognitive task.

Delivery of different genes into presynaptic and postsynaptic neocortical neurons connected by a BDNF-TrkB synapse.

Brain-Derived Neurotrophic Factor (BDNF) signaling through TrkB receptors has important roles in synapse formation, synaptic plasticity, learning, and specific diseases. However, it is challenging to relate BDNF-TrkB synapses to circuit physiology or learning, as BDNF-TrkB synapses are embedded in complex circuits that contain numerous neuron and synapse types. Thus, analyzing the physiology of neurons connected by BDNF-TrkB synapses would be advanced by a technology to deliver different genes into presynaptic and postsynaptic neurons, connected by a BDNF-TrkB synapse. Here, we report selective gene transfer across BDNF-TrkB synapses: The model system was the large projection from rat postrhinal to perirhinal cortex. The first gene transfer, into presynaptic neurons in postrhinal cortex, used a virus vector and standard gene transfer procedures. This vector expresses a synthetic peptide neurotransmitter composed of three domains, a dense core vesicle sorting domain, BDNF, and the His tag. Upon release, this peptide neurotransmitter binds to TrkB receptors on postsynaptic neurons. The second gene transfer, into connected postsynaptic neurons in perirhinal cortex, uses antibody-mediated, targeted gene transfer and an anti-His tag antibody, as the synthetic peptide neurotransmitter contains the His tag. Confocal microscope images showed that using untargeted gene transfer, only 10-15% of the transduced presynaptic axons were proximal to a transduced postsynaptic dendrite. But using targeted gene transfer, ∼70% of the transduced presynaptic axons were proximal to a transduced postsynaptic dendrite. This technology may support studies on the roles of neurons connected by BDNF-TrkB synapses in circuit physiology and learning.

Characteristic and intermingled neocortical circuits encode different visual object discriminations.

Synaptic plasticity and neural network theories hypothesize that the essential information for advanced cognitive tasks is encoded in specific circuits and neurons within distributed neocortical networks. However, these circuits are incompletely characterized, and we do not know if a specific discrimination is encoded in characteristic circuits among multiple animals. Here, we determined the spatial distribution of active neurons for a circuit that encodes some of the essential information for a cognitive task. We genetically activated protein kinase C pathways in several hundred spatially-grouped glutamatergic and GABAergic neurons in rat postrhinal cortex, a multimodal associative area that is part of a distributed circuit that encodes visual object discriminations. We previously established that this intervention enhances accuracy for specific discriminations. Moreover, the genetically-modified, local circuit in POR cortex encodes some of the essential information, and this local circuit is preferentially activated during performance, as shown by activity-dependent gene imaging. Here, we mapped the positions of the active neurons, which revealed that two image sets are encoded in characteristic and different circuits. While characteristic circuits are known to process sensory information, in sensory areas, this is the first demonstration that characteristic circuits encode specific discriminations, in a multimodal associative area. Further, the circuits encoding the two image sets are intermingled, and likely overlapping, enabling efficient encoding. Consistent with reconsolidation theories, intermingled and overlapping encoding could facilitate formation of associations between related discriminations, including visually similar discriminations or discriminations learned at the same time or place.

Neurons can be labeled with unique hues by helper virus-free HSV-1 vectors expressing Brainbow.

BACKGROUND: A central problem in neuroscience is elucidating synaptic connections, the connectome. Because mammalian forebrains contain many neurons, labeling specific neurons with unique tags is desirable. A novel technology, Brainbow, creates hundreds of hues by combinatorial expression of multiple fluorescent proteins (FPs). NEW METHOD: We labeled small numbers of neurons, and their axons, with unique hues, by expressing Brainbow from a helper virus-free Herpes Simplex Virus (HSV-1) vector. RESULTS: The vector expresses a Brainbow cassette containing four FPs from a glutamatergic-specific promoter. Packaging HSV-Brainbow produced arrays of seven to eight Brainbow cassettes, and using Cre, each FP gene was in a position to be expressed, in different cassettes. Delivery into rat postrhinal (POR) cortex or hippocampus labeled small numbers of neurons with different, often unique, hues. An area innervated by POR cortex, perirhinal (PER) cortex, contained axons with different hues. Specific axons in PER cortex were matched to specific cell bodies in POR cortex, using hue. COMPARISON WITH EXISTING METHODS: HSV-Brainbow is the only technology for labeling small numbers of neurons with unique hues. In Brainbow mice, many neurons contain the same hue. Brainbow-adeno-associated virus vectors require transduction of the same neuron with multiple vector particles, confounding neuroanatomical studies. Replication-competent Brainbow-pseudorabies virus vectors label multiple neurons with the same hue. CONCLUSIONS: Attractive properties of HSV-Brainbow include each vector particle contains multiple cassettes, representing numerous hues, recombination products are stabile, and experimental control of the number of labeled neurons. Labeling neurons with unique hues will benefit mapping forebrain circuits.

Targeted gene transfer of different genes to presynaptic and postsynaptic neocortical neurons connected by a glutamatergic synapse.

Genetic approaches to analyzing neuronal circuits and learning would benefit from a technology to first deliver a specific gene into presynaptic neurons, and then deliver a different gene into an identified subset of their postsynaptic neurons, connected by a specific synapse type. Here, we describe targeted gene transfer across a neocortical glutamatergic synapse, using as the model the projection from rat postrhinal to perirhinal cortex. The first gene transfer, into the presynaptic neurons in postrhinal cortex, used a virus vector and standard gene transfer procedures. The vector expresses an artificial peptide neurotransmitter containing a dense core vesicle targeting domain, a NMDA NR1 subunit binding domain (from a monoclonal antibody), and the His tag. Upon release, this peptide neurotransmitter binds to NMDA receptors on the postsynaptic neurons. Antibody-mediated targeted gene transfer to these postsynaptic neurons in perirhinal cortex used a His tag antibody, as the peptide neurotransmitter contains the His tag. Confocal microscopy showed that with untargeted gene transfer, ~3% of the transduced presynaptic axons were proximal to a transduced postsynaptic dendrite. In contrast, with targeted gene transfer, ≥ 20% of the presynaptic axons were proximal to a transduced postsynaptic dendrite. Targeting across other types of synapses might be obtained by modifying the artificial peptide neurotransmitter to contain a binding domain for a different neurotransmitter receptor. This technology may benefit elucidating how specific neurons and subcircuits contribute to circuit physiology, behavior, and learning.

CaMKII, MAPK, and CREB are coactivated in identified neurons in a neocortical circuit required for performing visual shape discriminations.

Current theories postulate that the essential information for specific cognitive tasks is widely dispersed in multiple forebrain areas. Nonetheless, synaptic plasticity and neural network theories hypothesize that activation of specific signaling pathways, in specific neurons, modifies synaptic strengths, thereby encoding essential information for performance in localized circuits. Consistent with these latter theories, we have shown that gene transfer of a constitutively active protein kinase C into several hundred glutamatergic and GABAergic neurons in rat postrhinal cortex enhances choice accuracy in visual shape discriminations, and the genetically-modified circuit encodes some of the essential information for performance. However, little is known about the role of specific signaling pathways required for learning, in specific neurons within a critical circuit. Here we show that three learning-associated signaling pathways are coactivated in the transduced neurons during both learning and performance. After gene transfer, but before learning a new discrimination, the calcium/calmodulin-dependent protein kinase (CaMKII), MAP kinase, and CREB pathways were inactive. During learning, these three pathways were coactivated in the transduced neurons. During later performance of the discrimination, CaMKII activity declined, but MAP kinase and CREB activity persisted. Because the transduced neurons are part of a circuit that encodes essential information for performance, activation of these learning-associated signaling pathways, in these identified neurons, is likely important for both learning and performance.

Overexpression of either lysine-specific demethylase-1 or CLOCK, but not Co-Rest, improves long-term expression from a modified neurofilament promoter, in a helper virus-free HSV-1 vector system.

Long-term expression from helper virus-free Herpes Simplex Virus (HSV-1) vectors is required for many specific neural gene therapies and studies on neuronal physiology. We previously developed a promoter that supports long-term, neuron-specific expression by fusing the chicken ß-globin insulator (INS), followed by an upstream enhancer from the rat tyrosine hydroxylase (TH) promoter, to a neurofilament heavy gene (NFH) promoter. Here, we examined the capability of specific transcription factors to further improve long-term expression from this promoter. Following a HSV-1 virus infection, the virus genome is localized to promyelocytic leukemia protein (PML) nuclear bodies (NB). At these sites, specific cellular transcription factors interact with HSV-1 encoded transcription factors, and together regulate HSV-1 gene expression. Importantly, lysine-specific demethylase-1 (LSD1), CLOCK, and Co-Rest each activate HSV-1 gene expression. However, gene expression from HSV-1 vectors differs in a number of important aspects from the virus, including no HSV-1 genes are expressed. Nonetheless, these observations raise the possibility that specific transcription factors may improve long-term expression from specific promoters in HSV-1 vectors. Here, we show that overexpression of either LSD1 or CLOCK improves long-term expression from the INS-TH-NFH promoter, but overexpression of Co-Rest supports levels of long-term expression similar to those supported by a control vector. Further, overexpression of LSD1 is compatible with neuron-specific expression. Thus, overexpressing specific transcription factors can improve long-term expression from specific cellular promoters in HSV-1 vectors, and the chromatin structure of the vector has an important role in enabling expression.

A 16 bp upstream sequence from the rat tyrosine hydroxylase promoter supports long-term expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system.

Helper virus-free Herpes Simplex Virus vector-mediated gene transfer has supported studies on neuronal physiology, and may support specific gene therapies. Long-term, neuron-specific expression is required for many of these applications. A neurofilament heavy gene (NFH) promoter does not support long-term expression. We previously developed a promoter that supports long-term expression by fusing 6.3 kb of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a NFH promoter, and this promoter has supported physiological studies. The TH promoter fragment contains an enhancer, as it has activity in both orientations and at a distance from the basal promoter. Identifying this enhancer may support further improvements in long-term expression. A previous deletion analysis identified two ~100 bp fragments that each support long-term expression, and are contained within an ~320 bp fragment located ~3 kb from the TH promoter transcription start site. As this analysis used overlapping fragments, the two ~100 bp fragments contained 44 or 23 bp of unique sequence. Here, we used mutagenesis to identify a short sequence that supports long-term expression. We studied a 42 bp sequence, centered on the 23 bp unique sequence. Analysis of the wt sequence, and five mutations containing clustered changes that spanned the sequence, identified two adjacent mutations that do not support long-term expression, which together defined a 16 bp maximum essential sequence. This 16 bp sequence contains a putative E2F-1/DP-1 transcription factor binding site, and this transcription factor is expressed in many brain areas.

Antibody-mediated targeted gene transfer of helper virus-free HSV-1 vectors to rat neocortical neurons that contain either NMDA receptor 2B or 2A subunits.

Because of the numerous types of neurons in the brain, and particularly the forebrain, neuron type-specific expression will benefit many potential applications of direct gene transfer. The two most promising approaches for achieving neuron type-specific expression are targeted gene transfer to a specific type of neuron and using a neuron type-specific promoter. We previously developed antibody-mediated targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors by modifying glycoprotein C (gC) to replace the heparin binding domain, which mediates the initial binding of HSV-1 particles to many cell types, with the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. We showed that a chimeric gC-ZZ protein is incorporated into vector particles and binds IgG. As a proof-of-principle for antibody-mediated targeted gene transfer, we isolated complexes of these vector particles and an anti-NMDA NR1 subunit antibody, and demonstrated targeted gene transfer to neocortical cells that contain NR1 subunits. However, because most forebrain neurons contain NR1, we obtained only a modest increase in the specificity of gene transfer, and this targeting specificity is of limited utility for physiological experiments. Here, we report efficient antibody-mediated targeted gene transfer to NMDA NR2B- or NR2A-containing cells in rat postrhinal cortex, and a neuron-specific promoter further restricted recombinant expression to neurons. Of note, because NR2A-containing neurons are relatively rare, these results show that antibody-mediated targeted gene transfer with HSV-1 vectors containing neuron type-specific promoters can restrict recombinant expression to specific types of forebrain neurons of physiological significance.

The vesicular glutamate transporter-1 upstream promoter and first intron each support glutamatergic-specific expression in rat postrhinal cortex.

Multiple applications of direct gene transfer into neurons require restricting expression to glutamatergic neurons, or specific subclasses of glutamatergic neurons. Thus, it is desirable to develop and analyze promoters that support glutamatergic-specific expression. The three vesicular glutamate transporters (VGLUTs) are found in different populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported on a plasmid (amplicon) Herpes Simplex Virus vector that contains a VGLUT1 promoter. This vector supports long-term expression in VGLUT1-containing glutamatergic neurons in rat postrhinal (POR) cortex, but does not support expression in VGLUT2-containing glutamatergic neurons in the ventral medial hypothalamus. This VGLUT1 promoter contains both the VGLUT1 upstream promoter and the VGLUT1 first intron. In this study, we begin to isolate and analyze the glutamatergic-specific regulatory elements in this VGLUT1 promoter. We show that the VGLUT1 upstream promoter and first intron each support glutamatergic-specific expression. We isolated a small, basal VGLUT1 promoter that does not support glutamatergic-specific expression. Next, we fused either the VGLUT1 upstream promoter or the first intron to this basal promoter. The VGLUT1 upstream promoter or the first intron, fused to the basal promoter, each supported glutamatergic-specific expression in POR cortex.

Genetic labeling of both the axons of transduced, glutamatergic neurons in rat postrhinal cortex and their postsynaptic neurons in other neocortical areas by herpes simplex virus vectors that coexpress an axon-targeted ß-galactosidase and wheat germ agglutinin from a vesicular glutamate transporter-1 promoter.

Neuronal circuits comprise the foundation for neuronal physiology and synaptic plasticity, and thus for consequent behaviors and learning, but our knowledge of neocortical circuits is incomplete. Mapping neocortical circuits is a challenging problem because these circuits contain large numbers of neurons, a high density of synapses, and numerous classes and subclasses of neurons that form many different types of synapses. Expression of specific genetic tracers in small numbers of specific subclasses of neocortical neurons has the potential to map neocortical circuits. Suitable genetic tracers have been established in neurons in subcortical areas, but application to neocortical circuits has been limited. Enabling this approach, Herpes Simplex Virus (HSV-1) plasmid (amplicon) vectors can transduce small numbers of neurons in a specific neocortical area. Further, expression of a particular genetic tracer can be restricted to specific subclasses of neurons; in particular, the vesicular glutamate transporter-1 (VGLUT1) promoter supports expression in VGLUT1-containing glutamatergic neurons in rat postrhinal (POR) cortex. Here, we show that expression of an axon-targeted ß-galactosidase (ß-gal) from such vectors supports mapping specific commissural and associative projections of the transduced neurons in POR cortex. Further, coexpression of wheat germ agglutinin (WGA) and an axon-targeted ß-gal supports mapping both specific projections of the transduced neurons and identifying specific postsynaptic neurons for the transduced neurons. The neocortical circuit mapping capabilities developed here may support mapping specific neocortical circuits that have critical roles in cognitive learning.

Identified circuit in rat postrhinal cortex encodes essential information for performing specific visual shape discriminations.

Learning theories hypothesize specific circuits encode essential information for performance. For simple tasks in invertebrates and mammals, the essential circuits are known, but for cognitive functions, the essential circuits remain unidentified. Here, we show that some essential information for performing a choice task is encoded in a specific circuit in a neocortical area. Rat postrhinal (POR) cortex is required for visual shape discriminations, protein kinase C (PKC) pathways mediate changes in neuronal physiology that support learning, and specific PKC genes are required for multiple learning tasks. We used direct gene transfer of a constitutively active PKC to prime a specific POR cortex circuit for learning visual shape discriminations. In the experiment, rats learned a discrimination, received gene transfer, learned new discriminations, received a small lesion that ablated approximately 21% of POR cortex surrounding the gene transfer site, and were tested for performance for discriminations learned either before or after gene transfer. Lesions of the genetically targeted circuit selectively interfered with performance for discriminations learned after gene transfer. Activity-dependent gene imaging confirmed increased activity in the genetically targeted circuit during learning and showed the essential information was sparse-coded in approximately 500 neurons in the lesioned area. Wild-type rats contained circuits with similar increases in activity during learning, but these circuits were located at unpredictable, different positions in POR cortex. These results establish that some essential information for performing specific visual discriminations can be encoded in a small, identified, neocortical circuit and provide a foundation for characterizing the circuit and essential information.

Antibody-mediated targeted gene transfer to NMDA NR1-containing neurons in rat neocortex by helper virus-free HSV-1 vector particles containing a chimeric HSV-1 glycoprotein C-staphylococcus A protein.

Because of the heterogeneous cellular composition of the brain, and especially the forebrain, cell type-specific expression will benefit many potential applications of direct gene transfer. The two prevalent approaches for achieving cell type-specific expression are using a cell type-specific promoter or targeting gene transfer to a specific cell type. Targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors modifies glycoprotein C (gC) to replace the heparin binding domain, which binds to many cell types, with a binding activity for a specific cell surface protein. We previously reported targeted gene transfer to nigrostriatal neurons using chimeric gC-glial cell line-derived neurotrophic factor or gC-brain-derived neurotrophic factor protein. Unfortunately, this approach is limited to cells that express the cognate receptor for either neurotrophic factor. Thus, a general strategy for targeting gene transfer to many different types of neurons is desirable. Antibody-mediated targeted gene transfer has been developed for targeting specific virus vectors to specific peripheral cell types; a specific vector particle protein is modified to contain the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. Here, we report antibody-mediated targeted gene transfer of HSV-1 vectors to a specific type of forebrain neuron. We constructed a chimeric gC-ZZ protein, and showed this protein is incorporated into vector particles and binds Ig G. Complexes of these vector particles and an antibody to the NMDA receptor NR1 subunit supported targeted gene transfer to NR1-containing neocortical neurons in the rat brain, with long-term (2 months) expression.

A helper virus-free HSV-1 vector containing the vesicular glutamate transporter-1 promoter supports expression preferentially in VGLUT1-containing glutamatergic neurons.

Multiple potential uses of direct gene transfer into neurons require restricting expression to specific classes of glutamatergic neurons. Thus, it is desirable to develop vectors containing glutamatergic class-specific promoters. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported a plasmid (amplicon) Herpes Simplex Virus (HSV-1) vector that placed the Lac Z gene under the regulation of the VGLUT1 promoter (pVGLUT1lac). Using helper virus-free vector stocks, we showed that this vector supported approximately 90% glutamatergic neuron-specific expression in postrhinal (POR) cortex, in rats sacrificed at either 4 days or 2 months after gene transfer. We now show that pVGLUT1lac supports expression preferentially in VGLUT1-containing glutamatergic neurons. pVGLUT1lac vector stock was injected into either POR cortex, which contains primarily VGLUT1-containing glutamatergic neurons, or into the ventral medial hypothalamus (VMH), which contains predominantly VGLUT2-containing glutamatergic neurons. Rats were sacrificed at 4 days after gene transfer, and the types of cells expressing ss-galactosidase were determined by immunofluorescent costaining. Cell counts showed that pVGLUT1lac supported expression in approximately 10-fold more cells in POR cortex than in the VMH, whereas a control vector supported expression in similar numbers of cells in these two areas. Further, in POR cortex, pVGLUT1lac supported expression predominately in VGLUT1-containing neurons, and, in the VMH, pVGLUT1lac showed an approximately 10-fold preference for the rare VGLUT1-containing neurons. VGLUT1-specific expression may benefit specific experiments on learning or specific gene therapy approaches, particularly in the neocortex.

Improved long-term expression from helper virus-free HSV-1 vectors packaged using combinations of mutated HSV-1 proteins that include the UL13 protein kinase and specific components of the VP16 transcriptional complex.

BACKGROUND: Herpes Simplex Virus (HSV-1) gene expression is thought to shut off recombinant gene expression from HSV-1 vectors; however, in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. These results raise the paradox that recombinant gene expression remains short-term even in the absence of almost all (approximately 99%) of the HSV-1 genome, HSV-1 genes, and HSV-1 gene expression. To resolve this paradox, we hypothesized that specific proteins in the HSV-1 virus particle shut off recombinant gene expression. In two earlier studies, we examined the effects on recombinant gene expression of packaging vectors using specific mutated HSV-1 proteins. We found that vectors packaged using mutated UL13 (a protein kinase), or VP16, or UL46 and/or UL47 (components of the VP16 transcriptional complex) supported improved long-term expression, and vectors packaged using mutated UL46 and/or UL47 also supported improved gene transfer (numbers of cells at 4 days). These results suggested the hypothesis that specific proteins in the HSV-1 particle act by multiple pathways to reduce recombinant gene expression. To test this hypothesis, we examined combinations of mutated proteins that included both UL13 and specific components of the VP16 transcriptional complex. RESULTS: A HSV-1 vector containing a neuronal-specific promoter was packaged using specific combinations of mutated proteins, and the resulting vector stocks were tested in the rat striatum. For supporting long-term expression, the preferred combination of mutated HSV-1 proteins was mutated UL13, UL46, and UL47. Vectors packaged using this combination of mutated proteins supported a higher efficiency of gene transfer and high levels expression for 3 months, the longest time examined. CONCLUSION: Vector particles containing this combination of mutated HSV-1 proteins improve recombinant gene expression. Implications of these results for strategies to further improve long-term expression are discussed. Moreover, long-term expression will benefit specific gene therapy applications.

Improved spatial learning in aged rats by genetic activation of protein kinase C in small groups of hippocampal neurons.

Age-related decline in human cognition is well known, and there are correlative changes in the function of neocortical and hippocampal neurons. Similarly, age-related decline in learning has been observed in rodents, including deficits in a hippocampal-dependent learning paradigm, the Morris water maze. Furthermore, there are correlative deficits in specific signaling pathways, including protein kinase C (PKC) pathways, in cerebellar, hippocampal, or neocortical neurons. PKC pathways are strong candidates for mediating the molecular changes that underlie spatial learning, as they play critical roles in neurotransmitter release and synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), and deletion of specific PKC genes results in deficits in learning. Conversely, genetic activation of PKC pathways in small groups of hippocampal or cortical neurons enhances learning in specific paradigms. In this study, the authors delivered a constitutively active PKC into small groups of hippocampal dentate granule neurons in aged rats (using a herpes simplex virus-1 vector). Aged 2-year-old rats that received the constitutively active PKC displayed improved performance in the Morris water maze relative to controls in three different measures. These results indicate that PKC pathways play an important role in mediating spatial learning in aged rats. Additionally, these results represent a system for studying the neural mechanisms underlying aging-related learning deficits, and potentially developing gene therapies for cognitive and age-related deficits.