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

Abstract

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.

1. Introduction

Specific behaviors and learning tasks are encoded by changing the strengths of specific synapses within complex, distributed circuits composed of many neuron and synapse types (Dudai, 1989; Rumelhart, McClelland and Group, 1986; Squire and Kandel, 2008). Further, forebrain areas contain hundreds, and possibly thousands, of neuron types, and these neurons make hundreds to thousands of synapses (Dudai, 1989). Genetic strategies have tremendous potential for understanding these processes. These strategies include optogenetics, or inhibition or activation of a specific transcriptional or signaling pathway (Dymecki and Kim, 2007; Fenno, Yizhar and Deisseroth, 2011; Luo, Callaway and Svoboda, 2008; Zhang, Wang, Kong, Lu, Lee, Liu et al., 2005). Currently, these strategies typically affect an entire circuit, or a large part of a circuit. Thus, understanding how specific neuron types, connected by a specific synapse type, support circuit physiology, behaviors, and learning would benefit from a technology that can deliver different genes selectively into the preand post-synaptic neurons that are connected by a specific synapse type.

Established genetic technologies for mapping and/or modifying circuits are sophisticated and powerful, but lack this capability. mGRASP has been used to visualize the synapses that connect specific neurons; however, mGRASP is not synapse type-specific and does not deliver genes selectively into the connected neurons, as it uses untargeted gene transfer (Feng, Kwon, Lee, Oh and Kim, 2014; Kim, Zhao, Petralia, Yu, Peng, Myers et al., 2011). Specific viruses, or genetically modified viruses, have been used to map anterograde or retrograde connections, across one or multiple synapses (reviewed in (Lo and Anderson, 2011)), but these technologies deliver the same gene into all the infected neurons. A technology based on Rabies Virus vectors delivers a gene into specific postsynaptic neurons, and then delivers a different gene into all the presynaptic neurons (Osakada, Mori, Cetin, Marshel, Virgen and Callaway, 2011); however, this technology requires a specific transgenic mouse, lacks synapse type specificity, and physiological experiments are limited to ~5–11 days after transduction, as Rabies Virus is neurotoxic.

We previously established a technology to deliver different genes into presynaptic and postsynaptic neurons connected by a specific synapse type (Zhang, Zhao, Cao, Li and Geller, 2012). The presynaptic vector expresses a synthetic peptide neurotransmitter that contains a dense core vesicle (DCV) sorting domain to ensure the peptide is processed as a peptide neurotransmitter, a receptor binding domain, and the His tag. Upon release, the synthetic peptide neurotransmitter binds to its cognate receptor on the postsynaptic neurons. Next, a gene is selectively delivered into these postsynaptic neurons, in a specific brain area; this gene transfer uses antibody-mediated, targeted gene transfer and anti-His tag antibody, as the synthetic peptide neurotransmitter contains the His tag (Zhang, Zhao, Cao, Li and Geller, 2012). We developed this technology using a synthetic peptide neurotransmitter designed to bind to NMDA NR1 subunits, present at most excitatory, glutamatergic synapses. For the model system, we studied the large glutamatergic projection from postrhinal (POR) cortex neurons to specific neurons in perirhinal (PER) cortex (Burwell and Amaral, 1998a). This model system was chosen as these neurons, and this synapse, are part of a large distributed neocortical circuit that can encode visual object discrimination learning, and we established a genetic intervention in POR cortex that enables the genetically-modified circuit to encode some of the essential information for this learning (Murray, Bussey and Saksida, 2007; Zhang, Cao, Kong, O’Brien, Baughns, Jan et al., 2010; Zhang, Zhao, Choi, Svestka, Wang, Nagayach et al., 2019). Thus, technology established in this system can be applied to other circuits that support synaptic plasticity, behaviors, and learning. Of note, in our initial study, we obtained only ~20 % gene transfer to connected neurons (Zhang, Zhao, Cao, Li and Geller, 2012). This limited level of specificity may not be sufficient to support specific physiological studies.

Here, we improved specific aspects of the technology to obtain ~80 % gene transfer to connected neurons, using the same synthetic peptide neurotransmitter and model system in the rat brain. This much higher level of specificity should enable physiological studies.

2. Materials and Methods

2.1. Vectors

pVGLUT1dcv-secretogranin/anti-NR1–10aa/his-tag has been described (Zhang, Zhao, Cao, Li and Geller, 2012). For glutamatergic-specific expression, the vector contains a mouse vesicular glutamate transporter-1 (VGLUT1) promoter. This promoter has both an upstream promoter fragment (7 kb) and the first intron (4.6 kb) (Rasmussen, Kong, Zhang, Liu, Wang, Szabo et al., 2007), and controls expression of a synthetic peptide neurotransmitter. The synthetic peptide neurotransmitter contains three domains (Zhang, Zhao, Cao, Li and Geller, 2012): The first domain is the human Secretogranin II DCV sorting domain composed of the signal peptide and amino acids (aa) 1–41 (Courel, Vasquez, Hook, Mahata and Taupenot, 2008; Gerdes, Rosa, Phillips, Baeuerle, Frank, Argos et al., 1989). The second domain is a 10 aa peptide (CQQHYSTPPC) that binds to NMDA NR1 subunits; this peptide is derived from an anti-NR-1 monoclonal antibody (Moskal, Yamamoto and Colley, 2001; Moskal, Kuo, Weiss, Wood, O’Connor Hanson, Kelso et al., 2005). This domain is followed by a spacer (60 bp) that separates this receptor binding domain from the third domain, the His tag.

The two postsynaptic vectors, pINS-TH-NFHdendrite-gfp (Zhang, Zhao, Cao and Geller, 2012) and pINS-TH-NFHdendrite-PkcΔGG (Zhang, Zhao, Cao, Li and Geller, 2012), have been reported. These vectors contain an established, chimeric neuron-specific promoter, composed of an insulator (INS), an upstream enhancer from the tyrosine hydroxylase (TH) promoter, and a neurofilament heavy gene (NFH) promoter (Zhang, Wang, Yang, Sun, Zhang, Wang et al., 2000). This promoter (INS-TH-NFH promoter) supports ≥90 % neuron-specific expression in a number of forebrain areas, including specific neocortical areas (Zhang, Wang, Yang, Sun, Zhang, Wang et al., 2000). Dendrite-targeted GFP (Kameda, Furuta, Matsuda, Ohira, Nakamura, Hioki et al., 2008) is composed of GFP fused to two dendrite-targeting sequences, a basolateral (dendrite) membrane-sorting domain (from the low density lipoprotein receptor), and a myristoylation/palmitoylation site (from Fyn). In dendrite-targeted PkcΔGG, the GFP domain in dendrite-targeted GFP was replaced with a mutated rat PKC ßII catalytic domain; the point mutation abolishes enzyme activity (Song, Wang, Bak, During, Bryan, Ashe et al., 1998); PkcΔGG contains the flag tag at the N-terminus.

2.2. Cells and vector packaging

BHK21 and 2–2 cells were grown in Dulbecco’s Modified Minimal Essential Medium (DMEM) with 10 % FBS, 4 mM glutamine, and penicillin/streptomycin. Cultures were grown at 37 °C in the presence of 5 % CO2, and 100 % humidity. 2–2 cells were used for HSV-1 vector packaging; G418 (0.5 mg/ml) was present during the growth of these cells, but was removed before cells were plated for packaging; 2–2 cells express the HSV-1 immediate early 2 (IE 2) gene (Smith, Hardwicke and Sandri-Goldin, 1992). The presynaptic vector and the comparison postsynaptic vector (pINS-THNFHdendrite-PkcΔGG) were packaged into HSV-1 particles using our standard procedure (Fraefel, Song, Lim, Lang, Yu, Wang et al., 1996; Sun, Zhang, Yang, Yu and Geller, 1999), but using a modern lipid, Lipofectamine 3000 (for a 60 mm plate: 16 μl Lipofectamine 3000, 11 μl P3000, 1.8 μg of each of the five HSV-1 cosmids, 2.2 μg vector, 1.5 ml total volume of transfection mixture). The postsynaptic vector for gene transfer to connected neurons (pINS-TH-NFHdendrite-gfp) was packaged for antibody-mediated, targeted gene transfer; this packaging contained gC--ZZ, a chimeric HSV-1 glycoprotein C (gC) containing an immunoglobulin (Ig) G binding domain, the Staphylococcus A protein ZZ domain, and gBpK⁻, and a mutated gB which lacks the glycosaminoglycan binding domain that supports binding to many cell types (Cao, Zhang and Geller, 2010; Zhang, Zhao, Cao, Li and Geller, 2012). Two mouse anti-His tag antibodies (a 1:1 mixture of Qiagen 34650 and 34660, RRID:AB_2714179 and RRID:AB_2687898; 5 mg/ml total antibody during the vector binding procedure) were incubated with the vector particles (Zhang et al., 2012b). The numbers of infectious vector particles per ml (IVP/ml) in the resulting purified vector stocks were determined using confluent (late-log phase) cultures of BHK21 cells; 24 h after transduction, cells were fixed and immunocytochemistry was performed. We previously showed that the titers of vector genomes (VG/ml, PCR assay) for a number of vector stocks that supported antibody-mediated, targeted gene transfer, and the packaging efficiencies (VG/ml / IVP/ml), were similar to those for vector stocks prepared using standard packaging (Cao, Zhang and Geller, 2010; Cao, Zhang and Geller, 2011; Gao, Sun, Wang and Geller, 2007; Rasmussen, Kong, Zhang, Liu, Wang, Szabo et al., 2007; Yang, Zhang, Zhang, Sun, Wang and Geller, 2001; Zhang, Wang, Yang, Sun, Zhang, Wang et al., 2000). As the vectors used here are similar to these previously characterized vectors and contain the same promoters, we did not determine VG/ml on the vector stocks used here. The titers of the vector stocks used here are: pVGLUT1dcv-secretogranin/anti-NR1–10aa/his-tag 1.7 ×10⁶ IVP/ml, pINS-THNFHdendrite-gfp/gC–ZZ+anti-His tag 2.8×10⁶ IVP/ml, and pINS-TH-NFHdendrite-PkcΔGG 2.2×10⁶ IVP/ ml. Wild-type HSV-1 was not observed (<10 plaque forming units/ml) in any of the vector stocks used for this study.

2.3. Gene transfer

All animal care and experimental procedures were approved by the Louisiana State University Health Science Center New Orleans IACUC. Long-Evans rats (male, 200–220 g) were purchased from Charles River and maintained on a 12h/12h light-dark cycle; water and food were available ad libitum.

Stereotactic surgeries and injection coordinates were as previously established (Zhang, Wang, Yang, Sun, Zhang, Wang et al., 2000; Zhang, Zhao, Cao, Li and Geller, 2012). The key parameters were that rats were anesthetized using a Ketamine and Xylazine mixture (20 mg/ml, 2 mg/ml), at concentrations of 60 mg/kg and 6 mg/kg, and additional anesthesia was administered as indicated. The rats were inserted into a small rodent stereotactic frame. For the presynaptic gene transfer, we performed one injection into the left hemisphere of POR cortex; the injection coordinates were anterior-posterior (AP) −8.0, medial-lateral (ML) −6.0, and dorsal-ventral (DV) −5.2 (Paxinos and Watson, 1986; Zhang, Zhao, Cao, Li and Geller, 2012). To perform the postsynaptic gene transfer, a 1:1 (IVP:IVP) mixture of the experimental and comparison postsynaptic vectors was delivered by one injection into the left hemisphere of PER cortex; the injections coordinates were AP −5.0, ML −6.6, DV −7.0. For these injection coordinates, AP was measured from bregma, ML from the sagittal suture, and DV from the bregma-lambda plane. A micropump (model 100, KD Scientific) was used to perform these injections; 3 μl of vector stock was delivered over 5 minutes, the needle was left in place for an additional 5 minutes, and then the needle was slowly retracted.

2.4. Immunohistochemistry

After perfusions (Zhang, Wang, Yang, Sun, Zhang, Wang et al., 2000), a freezing microtome was used to prepare 25 μm coronal sections that contained POR and/or PER cortices; free-floating sections were used for immunofluorescent assays following our established protocol (Zhang, Wang, Yang, Sun, Zhang, Wang et al., 2000). The primary antibodies were rabbit anti-His tag (Cell Signaling RRID:AB_10691695, 1:200 dilution), and either mouse anti-GFP (ThermoFisher RRID:AB_2334927, 1:500 dilution) or mouse anti-flag (Sigma RRID:AB_259529, 1:500 dilution). The primary antibodies were visualized by using Texas Red-conjugated goat anti-rabbit IgG (Vector RRID:AB_2336199, 1:200 dilution) and FITC-conjugated goat anti-mouse IgG (ThermoFisher RRID:AB_2534753, 1:200 dilution). A negative control was omission of the primary antibodies.

A Leica TCS SP8 X confocal microscope was used to capture images. For each field that was analyzed, a single image was obtained in each of the two channels, thereby ensuring that processes that are proximal in the X and Y dimensions were also proximal in the Z dimension. We did not collect “image stacks” in the Z dimension. To quantify gene transfer to connected neurons, the two images that contained His tag-IR and either GFP-IR or flag-IR were merged, using Adobe Photoshop. The connected axons and dendrites were scored in these merged images. For each field that was examined, all the His tag-IR axons were scored for being proximal to, or distant from, either a GFP-IR or a flag-IR dendrite.

2.5. Statistical analyses

StatPlus was used to perform descriptive statistical analyses and one-way ANOVAs with α=0.05; the data were normally distributed, and p<0.05 was considered significant.

3. Results

3.1. The model system, and the strategy for targeting gene transfer to neurons that are connected by a specific synapse type

For the model system to enhance gene transfer to connected neurons, across excitatory, NMDA receptor-containing synapses, we used the large projection from POR cortex to PER cortex (Fig. 1) (Agster and Burwell, 2009; Burwell and Amaral, 1998a; Burwell and Amaral, 1998b; Burwell, 2000). This is the same model system that we used in our initial study on gene transfer to connected neurons, enabling direct and quantitative comparisons between the data obtained here to the data in our initial study (Zhang, Zhao, Cao, Li and Geller, 2012). For the first, presynaptic gene transfer, vectors were injected into POR cortex. For the second, postsynaptic gene transfer, vectors were injected into PER cortex, specifically at a site that receives projections from the transduced neurons in POR cortex.

Fig. 1.

The strategy to deliver different genes into pre- or post-synaptic neurons connected by a synapse that contains NMDA NR1 subunits. The model system was the synapses from projection neurons in POR cortex to specific neurons in PER cortex (Agster and Burwell, 2009). The first gene transfer, into the presynaptic neurons in POR cortex, uses standard gene transfer technology. The presynaptic vector expresses a synthetic peptide neurotransmitter from the VGLUT1 promoter, a glutamatergic-specific promoter. The synthetic peptide neurotransmitter is composed of three domains: i) a DCV sorting domain, ii) a NMDA NR-1 subunit-binding domain, to support binding to NMDA receptors on the postsynaptic neurons, and iii) the His tag. As shown in the right column of neurons, following gene transfer, synthetic peptide neurotransmitter (ellipse) is released from the axon terminals and binds to NMDA receptors (arc) that are present on specific postsynaptic neurons in PER cortex. The second, postsynaptic gene transfer is selective gene transfer for these neurons; this selectivity is obtained by antibody-mediated targeting. For antibody-mediated targeting, anti-His tag antibodies are bound to vector particles that contain a chimeric protein; the IgG binding domain from Staphylococcus A protein (ZZ domain) was fused to HSV-1 glycoprotein C (gC--ZZ+anti-His tag). This postsynaptic vector uses the pan-neuronal INS-TH-NFH promoter to express a dendrite-targeted GFP. This system supports gene transfer with high specificity for the connected postsynaptic neurons: As shown in the second postsynaptic neuron from the right, axon collaterals from transduced neurons will release the synthetic peptide neurotransmitter at other synapses, but if the postsynaptic neuron lacks NMDA receptors, that postsynaptic neuron will not be transduced. Additionally, as shown in the next column of neurons, postsynaptic neurons that express NMDA receptors, but do receive a synapse from an axon terminal of a transduced presynaptic neuron, will not be transduced. Moreover, ensembles of untransduced neurons connected to neurons lacking NMDA receptors will not be affected, as illustrated in the left column of neurons.

The strategy to deliver different genes into neurons connected by a specific synapse type contains three steps (Fig. 1): The first gene transfer, into the presynaptic neurons, employs standard gene transfer procedures. This vector expresses a synthetic peptide neurotransmitter, containing three domains: A domain that supports processing and release of the recombinant protein as a peptide neurotransmitter, a DCV sorting domain (Dikeakos and Reudelhuber, 2007), is the N-terminal domain. The middle domain is designed to bind to NMDA NR1 subunits on specific postsynaptic neurons, and contains a critical sequence from an anti-NMDA NR1 subunit antibody (Moskal, Yamamoto and Colley, 2001; Moskal, Kuo, Weiss, Wood, O’Connor Hanson, Kelso et al., 2005; Zhang, Zhao, Cao, Li and Geller, 2012). A His tag is the C-terminal domain. Upon release, the synthetic peptide neurotransmitter binds to postsynaptic neurons that contain its cognate receptor. The second gene transfer is selective for postsynaptic neurons that receive synapses from the transduced presynaptic neurons. This selectivity is conferred by antibody-mediated, targeted gene transfer (Cao, Zhang and Geller, 2010; Cao, Zhang and Geller, 2011) using an anti-His tag antibody, as the synthetic peptide neurotransmitter contains the His tag. Antibody-mediated, targeted gene transfer is achieved by fusing an IgG binding domain, the Staphylococcus A protein ZZ domain, to a protein on surface of HSV-1 vector particles, gC (gC--ZZ chimeric protein); anti-His tag antibody is bound to these vector particles prior to gene transfer.

This strategy is designed to achieve high specificity for synapse type (Fig. 2). The postsynaptic gene transfer is selective for neurons that both express a specific receptor and receive a synapse from a transduced presynaptic neuron. Proximal postsynaptic neurons that receive a synapse from the same transduced presynaptic neuron, but do not express the indicated receptor, will not be transduced. To generalize, postsynaptic neurons that receive a synapse from a transduced presynaptic neuron, but lack the indicated receptor, will not be transduced. Additionally, postsynaptic neurons that contain the indicated receptor, but lack a synapse from a transduced presynaptic neuron, will not be transduced. Further, while most of the transduced presynaptic neurons likely innervate multiple brain areas, only the postsynaptic neurons in one specific brain area will be transduced. Moreover, additional circuit and neuron specificity can be obtained by choosing appropriate promoters for the pre- and post-synaptic vectors. To summarize, in the forebrain, presynaptic neurons make hundreds to thousands of synapses with numerous postsynaptic neurons in multiple areas, but this strategy is designed to limit the postsynaptic gene transfer to a small subset of the postsynaptic neurons; specifically, the postsynaptic neurons located in one area and connected via a specific synapse type.

Fig. 2.

The transcription units in pVGLUT1dcv-secretogranin/anti-NR1–10aa/his-tag or the postsynaptic vectors. All of these vectors contain a standard HSV-1 vector backbone that enables packaging of vector DNA into HSV-1 particles (Zhang, Zhao, Cao, Li and Geller, 2012). The presynaptic vector contains the VGLUT1 promoter to support glutamatergic-specific expression of a synthetic peptide neurotransmitter that binds to NMDA NR1 subunits. This synthetic peptide neurotransmitter contains a DCV sorting domain from Secretogranin II, for processing the peptide as a peptide neurotransmitter, a 10 aa peptide that binds to NR1, derived from an antibody, and the His tag. The two postsynaptic vectors have been previously established; they use the pan-neuronal INSTH-NFH promoter to express either a dendrite-targeted GFP or PkcΔGG (an enzymatically inactive PKC). The two domains that support dendrite-targeting signals are a myristoylation/palmitoylation (Myr) site (from Fyn) and a dendrite/basolateral membrane-sorting domain (from the low density lipoprotein receptor) (Kameda, Furuta, Matsuda, Ohira, Nakamura, Hioki et al., 2008).

3.2. Vectors

Again, to enable direct and quantitative comparisons between the data obtained here to the data in our initial study, we used a synthetic peptide neurotransmitter, and corresponding presynaptic vector, that was reported in our initial study (Zhang, Zhao, Cao, Li and Geller, 2012). This synthetic peptide neurotransmitter contains three domains (Fig. 2). First, at the N-terminus, a DCV sorting domain from human Secretogranin II (Courel, Vasquez, Hook, Mahata and Taupenot, 2008; Gerdes, Rosa, Phillips, Baeuerle, Frank, Argos et al., 1989). Second, a 10 aa NMDA NR1-binding domain, derived from an antibody (Moskal, Yamamoto and Colley, 2001; Moskal, Kuo, Weiss, Wood, O’Connor Hanson, Kelso et al., 2005). Third, a His tag, the C-terminal domain.

As glu is the ligand for NMDA receptors, this synthetic peptide neurotransmitter was expressed from a glutamatergic-specific, VGLUT1 promoter (Rasmussen, Kong, Zhang, Liu, Wang, Szabo et al., 2007; Zhang and Geller, 2010) (Fig. 2). VGLUT1-containing glutamatergic neurons are the predominant glutamatergic neuron type in the neocortex, and previous studies established that this VGLUT1 promoter supports ≥90 % VGLUT1-specific expression in POR cortex from HSV-1 vectors. This vector was packaged into HSV-1 particles using standard helper virus-free packaging (Fraefel, Song, Lim, Lang, Yu, Wang et al., 1996), but using a modern lipid for the transfection reagent, Lipofectamine 3000, as detailed in the methods section.

Also, to enable direct and quantitative comparisons between the data obtained here to the data in our initial study, we used the same targeted and control postsynaptic vectors that were used in our initial study (Zhang, Zhao, Cao, Li and Geller, 2012). For marking the cell bodies and dendrites of the transduced postsynaptic neurons, the targeted vector expresses a dendrite-targeted GFP (Fig. 2) (Zhang, Zhao, Cao, Li and Geller, 2012). This construct contains two sequences that confer dendrite localization, a myristoylation/palmitoylation site and a basolateral (dendrite) membrane-sorting domain (Kameda, Furuta, Matsuda, Ohira, Nakamura, Hioki et al., 2008). This dendrite-targeted GFP was expressed from an established neuron-specific promoter; the INS-TH-NFH promoter supports ≥90 % neuron-specific expression in a number of forebrain areas, including POR and PER cortices, from HSV-1 vectors (Zhang, Wang, Yang, Sun, Zhang, Wang et al., 2000).

We packaged the vector that expresses a dendrite-targeted GFP for antibody-mediated, targeted gene transfer to neurons that have the His tag on their surface (Zhang, Zhao, Cao, Li and Geller, 2012). To enable antibody-mediated gene transfer, the IgG binding domain from Staphylococcus A protein, the ZZ domain, was fused to HSV-1 gC, as this glycoprotein is present on the surface of the vector particles (Cao, Zhang and Geller, 2010). The vector particles produced by this packaging were incubated with anti-His tag antibody, and these vector particle-antibody complexes were purified for use in gene transfer. Thus, these vector particle-antibody complexes will bind to the His tag in a synthetic peptide neurotransmitter, which is bound to the NR1 subunit on the surface of a postsynaptic neuron. Subsequently, the vector particles enter neurons using the same mechanisms that mediate entry by wt HSV-1 (Spear and Longnecker, 2003).

The comparison, or untargeted, postsynaptic vector expresses a different marker that is also dendrite-targeted. The dendrite-targeted GFP was modified to replace the GFP domain with an enzymatically inactive PKC catalytic domain, PkcΔGG (Fig. 2) (Song, Wang, Bak, During, Bryan, Ashe et al., 1998; Zhang, Zhao, Cao, Li and Geller, 2012). The resulting vector was packaged using the standard helper virus-free packaging procedure, in the presence of wt gC (Fraefel, Song, Lim, Lang, Yu, Wang et al., 1996).

3.3. Efficient delivery of different genes into pre- or post-synaptic neurons connected by a NMDA NR1-containing synapse

Moreover, to enable direct and quantitative comparisons between the data obtained here to the data in our initial study, we used the same experimental design as in our initial study (Zhang, Zhao, Cao, Li and Geller, 2012). First, the presynaptic vector was delivered into POR cortex, via stereotactic injection (one injection site in one hemisphere). Second, eight days later, a 1:1 mixture of the two postsynaptic vectors was injected into PER cortex; the area proximal to this injection site receives axons from the transduced neurons in POR cortex (Zhang, Cao, Li, Zhao and Geller, 2010; Zhang, Zhao, Cao, Li and Geller, 2012). Third, 4 days later, the rats were sacrificed. The eight-day period between the first and second surgeries supports the presynaptic gene transfer, expression and processing of the synthetic peptide neurotransmitter, transport to axon terminals, release, and binding to NR1 subunits on postsynaptic neurons. The four-day period between the second surgery and rat sacrifice supports the postsynaptic gene transfer, expression of the dendrite-targeted markers, and transport into dendrites. Injecting a mixture of the two postsynaptic vectors controls for variations in the injection sites among the rats; for each rat, adjacent sections were analyzed for targeted or untargeted gene transfer (dendrite-targeted GFP or dendrite-targeted PkcΔGG).

We observed expression of the synthetic peptide neurotransmitter in neurons in POR cortex, and confirmed that POR cortex lacks expression from the postsynaptic vectors, which were injected into a different brain area, PER cortex. As HSV-1 particles can be transported retrogradely in axons, significant levels of such transport could potentially complicate the interpretation of data on gene transfer to connected neurons. However, using the injection site in POR cortex, we previously observed only minimal amounts of retrograde transport of these vectors between neocortical areas (Zhang, Wang, Kong, Lu, Lee, Liu et al., 2005; Zhang, Cao, Li, Zhao and Geller, 2010). In this study, recombinant proteins were detected by immunofluorescent costaining, and a confocal microscope was used to obtain photomicrographs. Low power views show the position of a presynaptic injection sites in POR cortex (Fig. 3A) or postsynaptic injection sites in PER cortex (Fig. 3C), and higher power views showed transduced neurons proximal to each injection site (Fig 3 B and D). Further analysis showed that the area proximal to an injection site in POR cortex contained neuronal cell bodies that displayed expression of the synthetic peptide neurotransmitter from the pre-synaptic vector, which was injected into POR cortex (Fig. 4 A and D). In contrast, no expression was observed from either postsynaptic vector, as these vectors were injected into a different area, PER cortex (Fig. 4 B and E); no transduced cell bodies were observed from retrograde transport of this vector from axon terminals in PER cortex to cell bodies in POR cortex. The merge of each field show expression from only the presynaptic vector that was injected into POR cortex (Fig. 4 C and F). Omission of the primary antibodies resulted in no signal (Fig. 4G–I). For reasons detailed in the next paragraph, for each field and emission channel (fluorescent-conjugated antibody), we collected a single confocal image; we did not collect “image stacks”. Thus, these images captured cross sections of cell bodies at different angles, with a range of diameters, as a single confocal image has a small depth of field.

Fig. 3.

The locations of the pre- and post-synaptic injection sites in POR or PER cortex, and transduced neurons proximal to each injection site. (A and B) The presynaptic vector pVGLUT1dcvsecretogranin/anti-NR1–10aa/his-tag, His tag-IR; (A) a low power view showing that transduced neurons are located in POR cortex within a section, and (B) high power view of the box in panel A showing transduced neurons. (C and D) The postsynaptic vector pINS-TH-NFHdendrite-gfp/gC–ZZ+anti-His tag, GFP-IR; (C) a low power view showing that transduced neurons are located in PER cortex within a section, and (D) high power view of the box in panel C showing transduced neurons. Scale bars: (A and C) 1,200 μm; (B and D) 50 μm.

Fig. 4.

Recombinant proteins in POR cortex following injection of a presynaptic vector, pVGLUT1dcvsecretogranin/anti-NR1–10aa/his-tag, into POR cortex, and then coinjection of the two postsynaptic vectors into PER cortex. The presynaptic vector was delivered, and eight days later the two postsynaptic vectors (pINS-TH-NFHdendrite-gfp/gC–ZZ+anti-His tag, pINS-TH-NFHdendrite-PkcΔGG) were delivered (1:1 mixture); the rats were sacrificed four days later. Using immunofluorescent costaining, POR cortex was analyzed for recombinant proteins. (A-C) Expression from the presynaptic vector and the targeted postsynaptic vector pINS-TH-NFHdendrite-gfp/gC–ZZ+anti-His tag; (A) His tag-IR (presynaptic vector), (B) GFP-IR (targeted postsynaptic vector), and (C) merge. Arrows, neuronal cell bodies that contain the synthetic peptide neurotransmitter. (D-F) Costaining for expression from the presynaptic vector and pINS-TH-NFHdendrite-PkcΔGG; (D) His tag-IR, (E) flag-IR (comparison postsynaptic vector; flag is fused to PkcΔGG), and (F) merge. (G-I) The presynaptic vector and the targeted postsynaptic vector pINS-TH-NFHdendrite-gfp/gC–ZZ+anti-His tag, but omission of the primary antibodies; (G) His tag-IR (presynaptic vector), (H) GFP-IR (targeted postsynaptic vector), and (I) merge. Scale bar: 50 μm.

We assayed gene transfer to connected neurons in PER cortex. For alternating sections, we covisualized axon terminals containing the synthetic peptide neurotransmitter and postsynaptic dendrites and cell bodies containing either the targeted or comparison postsynaptic marker (dendrite-targeted GFP or dendrite-targeted PkcΔGG). For each field and emission channel (detects one antibody), we obtained a single confocal image, which has a depth of field of ~1 μm. Thus, axons and dendrites proximal in the X and Y dimensions (height and width) will also be proximal in the Z dimension (depth). However, given this limited depth of field, most axons or dendrites are shown in either cross section or oblique section, and the images contain limited lengths of these processes. For this synthetic peptide neurotransmitter and the targeted postsynaptic vector, we observed many transduced axons proximal to a transduced dendrite, but some of the transduced axons were distant from a transduced dendrite (Fig. 5A–D). In the high power view in Fig. 5D, three examples are labeled of a transduced axon (arrows) proximal to a transduced dendrite (arrowheads), and one example is labeled of a transduced axon distant from a transduced dendrite. Depending upon the orientation of the synapse relative to the plane of the photomicrographs, some connected synapses show the axon and dendrite overlapping, and other synapses show the axon and dendrite proximal to each other. In contrast, for this same synthetic peptide neurotransmitter and the comparison, untargeted postsynaptic vector, most of the transduced axons were distant from transduced dendrites (Fig. 5E–H). In the high power view in Fig. 5H, three examples are labeled of a transduced axon (arrows) distant from a transduced dendrite (arrowheads), and one example is labeled of a transduced axon proximal to a transduced dendrite. To quantify this data, for the synthetic peptide neurotransmitter, and either the targeted or comparison postsynaptic marker, we counted the numbers of transduced axons adjacent to, or distant from, a transduced dendrite or cell body. These counts (Table 1) showed that the combination of this synthetic peptide neurotransmitter and the targeted postsynaptic marker supported high levels, 80 %, of gene transfer to connected neurons; in contrast, this synthetic peptide neurotransmitter and the comparison, untargeted postsynaptic marker supported only a low level of gene transfer to connected neurons, 22 % (targeted vs. untargeted postsynaptic marker, F_1,4 = 300 p<0.001, 1-way ANOVA). Our initial study used the same synthetic peptide neurotransmitter, pre- and post-synaptic vectors, model system, and experimental design, but different times for performing the experiments, and different confocal microscopes. In this initial study, we observed only 20±1 % gene transfer to connected neurons (Zhang, Zhao, Cao, Li and Geller, 2012), whereas in the present study we obtained 80+2 % gene transfer to connected neurons (Table 1). Thus, by improving the technology we achieved a significant improvement in the efficiency of gene transfer to connected neurons, and the data in both studies have low variances. These differences are statistically significant (F_1,4 = 499 p<0.001), but this statistical analysis should be interpreted cautiously, due to the similarities and differences between the two studies, as just noted.

Fig. 5.

Efficient gene transfer to connected neurons using the synthetic peptide neurotransmitter dcvsecretogranin/anti-NR1–10aa/his-tag. The presynaptic vector was injected into POR cortex, and eight days later the two postsynaptic vectors were coinjected into PER cortex. The rats were sacrificed four days later. Using immunofluorescent costaining, alternating sections in PER cortex were analyzed for expression from the presynaptic vector and either the targeted or comparison postsynaptic vector. (A-D) The presynaptic vector, and the targeted postsynaptic vector, support efficient gene transfer to connected neurons; (A) His tag-IR (presynaptic vector), (B) GFP-IR (targeted postsynaptic vector), (C) merge, and (D) merge, an enlarged view of the area within the rectangle in (C). This area contains several transduced axons that are proximal to a transduced dendrite. Arrows, transduced axons; arrowheads, transduced dendrites. (E-H) The presynaptic vector, and the untargeted, control postsynaptic vector support low levels of gene transfer to connected neurons; (E) His tag-IR, (F) flag-IR (control postsynaptic vector), (G) merge, and (H) merge, an enlarged view of the area within the rectangle in (G). Within this area, most His tag-IR axons are distant from flag-IR dendrites. Scale bars: (A-C and E-G) 50 μm, and (D and H) 50 μm.

Table 1.

Targeted gene transfer across NMDA NR1-containing synapses; the percentage of transduced presynaptic axons proximal to a transduced postsynaptic dendrite

	Postsynaptic vector
Presynaptic	Targeting vector		Comparison vector
Vector	His-tag-IR	% GFP-IR	His-tag-IR	% Flag-IR
	axons	costaining	axons	costaining
pVGLUT1dcv-secretogranin/
anti-NR1–10aa/his-tag	620±98	80±2	635±98	22±2

In each section that was examined, all the transduced presynaptic axons were scored for being adjacent to, or distant from, a transduced postsynaptic dendrite. Three rats; mean±s.e.m. are shown.

4. Discussion

We previously developed a strategy and a technology to selectively deliver different genes into pre- or post-synaptic neurons connected by a specific type of synapse (Zhang, Zhao, Cao, Li and Geller, 2012). Our initial report used the same reagents, model system, and experimental design as used here, but achieved only ~20 % targeting to connected neurons (Zhang, Zhao, Cao, Li and Geller, 2012). Here, we used a modern lipid, Lipofectamine 3000, for the transfection for packaging, which increased the titers ~3 to 5-fold, and we obtained ~80 % targeting to connected neurons. As the transduction efficiency in the rodent brain increases non-linearly with the amount of injected vector, this modest increase the titers could account for the large, 4-fold increase in the targeting specificity, from 20 % to 80 %. Of note, as NR1 is present in most excitatory, glutamatergic synapses, the synthetic peptide neurotransmitter used here is specific for these synapses.

Using the methodology established here as the foundation, we recently developed new synthetic peptide neurotransmitters with different specificities. We changed the NR1 targeting domain to target gene transfer to receptors present in only specific subsets of glutamatergic synapses. We inserted BDNF to target to TrkB receptors, and we used ScFv that recognize either NR2B or mGluR5 (Nagayach, Singh and Geller, 2019a; Nagayach, Singh and Geller, 2019b; Nagayach, Singh and Geller, 2019c). These new synthetic peptide neurotransmitters were tested in the same model system used here, and supported 70 to 80 % targeting to each cognate receptor. Further, we inserted a ScFv that recognizes the GABAA receptor into a synthetic peptide neurotransmitter, and obtained 61 % specific gene transfer to connected interneurons in POR cortex (Nagayach, Singh, De Blas and Geller, 2019).

This strategy has significant generality. In addition to targeting gene transfer across specific synapse types, specificity for either the pre- or post-synaptic neuron type has been established. Here, the presynaptic gene transfer used standard, untargeted gene transfer, to optimize the procedure. However, the presynaptic gene transfer could be restricted to a specific neuron type; targeted gene transfer to GDNF receptor-, TrkB-, NR1-, NR2A-, or NR2B-expressing neurons has been established (Cao, Zhang and Geller, 2010; Cao, Zhang and Geller, 2011; Wang, Kong, Zhang, Sun and Geller, 2005). Additionally, here we used a pan-neuronal promoter, as pan-neuronal expression of the postsynaptic vector is useful for quantifying gene transfer to connected neurons. However, expression could be restricted to a specific postsynaptic neuron type by using a neuron type-specific promoter; glutamatergic- or GABAergic-specific promoters are established (Rasmussen, Kong, Zhang, Liu, Wang, Szabo et al., 2007). Moreover, this strategy is compatible with a number of other virus vector systems, as most virus vectors are capable of expressing a synthetic peptide neurotransmitter, and antibody-mediated, targeted gene transfer has been reported using a number of well-known virus vector systems, including Sindbis Virus, Adenovirus, Adeno-Associated Virus (AAV), Lentivirus, classical retrovirus, and HSV-1 vectors (Bergman, Whitaker-Dowling, Gao, Griffin and Watkins, 2003; Cao, Zhang and Geller, 2010; Morizono, Bristol, Xie, Kung and Chen, 2001; Morizono and Chen, 2005; Morizono, Xie, Ringpis, Johnson, Nassanian, Lee et al., 2005; Ohno, Sawai, Iijima, Levin and Meruelo, 1997; Ried, Girod, Leike, Buning and Hallek, 2002; Tai, Logg, Park, Anderson, Press and Kasahara, 2003; Volpers, Thirion, Biermann, Hussmann, Kewes, Dunant et al., 2003).

The capability to efficiently and selectively transduce the pre- and post-synaptic neurons that are connected by a specific synapse type should support novel studies on the roles of these neurons and synapses in synaptic plasticity, circuit physiology, behaviors, learning, and disorders caused by altered physiology at a specific synapse type. The particular pathways that mediate synaptic plasticity for a specific learning task might be identified by expressing a gene that modifies a pathway selectively in the connected, postsynaptic neurons; a siRNA or CRISPR/cas9 might be used to either block or activate a pathway.

Highlights.

• Gene transfer to neurons connected by a specific synapse type

• A synthetic peptide neurotransmitter that binds to a specific postsynaptic receptor

• Antibody-mediated, targeted gene transfer into the postsynaptic neurons

• 80 % specific gene transfer into connected neurons