Biology subjects

Wickman, K.

Publications and source records attributed to Wickman, K..

5 recordsLinked to original sources

bioRxiv2026

Nanobody-Functionalized AAV achieves Promoter-independent Neuronal Targeting in the CNS

Adeno-associated virus (AAV) vectors are widely used for gene delivery to the central nervous system, but natural capsid tropism is broad and cell-type restriction is typically imposed transcriptionally using promoters and enhancers that consume packaging capacity and often drive weak expression. Here, we engineer neuronal targeting directly into the AAV-DJ capsid by ablating its endogenous heparan sulfate proteoglycan (HSPG) affinity and genetically displaying a nanobody against the Group 1 metabotropic glutamate receptor mGluR5 within the VP1 subunit at a permissive capsid loop (T456), generating AAV-m5. Western blot confirmed incorporation of the nanobody-VP1 fusion into assembled capsids. In primary hippocampal neuron cultures, ablating HSPG binding abolished infectivity and nanobody display rescued transduction while restricting GFP expression almost exclusively to mGluR5-positive neurons. Heparin competition assays showed that, unlike wild-type AAV-DJ, AAV-m5 transduction was unaffected by exogenous heparin, confirming that entry occurs independently of HSPG binding. Packaged with a strong constitutive promoter (CAG), AAV-m5 achieved neuron-restricted expression comparable to or exceeding that of wild-type AAV-DJ driven by a neuron-specific promoter (hSyn) and produced negligible expression under an astrocyte-specific promoter (GFAP) despite promoter activity in glia, demonstrating that capsid-level targeting can substitute, or complement, transcriptional restriction. Following stereotactic injection into the mouse hippocampus, AAV-m5 achieved an eight-fold higher proportion of transduced neurons than wild-type AAV-DJ at equivalent titers. Delivery to Grm5-null hippocampus reduced both the intensity and the spatial extent of transduction, confirming that the broad hippocampal transduction achieved by AAV-m5 is mGluR5-dependent. Together, these results establish nanobody-functionalized AAV-DJ as a modular, single-component platform for precision CNS gene delivery that circumvents the packaging and expression trade-offs of promoter-based cell-type restriction, with potential for retargeting to additional CNS cell types and disease-relevant receptors.

synthetic biology↗

bioRxiv2026

AAV-only targeting of ventral tegmental area dopamine neurons for optical self-stimulation studies in mice

Studies employing optogenetic approaches in rodent models have highlighted the important contribution of ventral tegmental area (VTA) dopamine (DA) neurons to reward, learning, and motivation. Selective manipulation of VTA DA neurons is generally achieved in these studies using transgenic mouse or rat lines that express Cre recombinase under the control of a promoter active in DA neurons, combined with intra-VTA infusion of adeno-associated virus (AAV) vectors harboring Cre recombinase-dependent expression cassettes. Reliance on transgenic Cre driver lines is expensive and decreases study efficiency, and available driver lines have unique limitations. Here, we report the development of an AAV-only approach that permits genetic access to VTA DA neurons and can support optogenetic self-stimulation in mice. We used a 2.5 kb fragment of the mouse tyrosine hydroxylase promoter (mTH) to drive Cre expression in VTA DA neurons. Intra-VTA co-infusion of AAV8-mTH-Cre with an AAV vector harboring a Cre-dependent yellow fluorescent protein expression cassette yielded high efficiency (82%) and high fidelity (73%) targeting of tyrosine hydroxylase-positive VTA neurons in C57BL/6J mice. Co-infusion of AAV8-mTH-Cre with a vector harboring a Cre-dependent channelrhodopsin (ChR2) expression cassette permitted optical regulation of VTA neurons with electrophysiological features consistent with VTA DA neurons. Moreover, C57BL/6J mice expressing ChR2 in VTA DA neurons rapidly acquired optical self-stimulation behavior. Thus, this AAV-only approach should facilitate investigation of VTA DA neuron contributions to reward-related behaviors and permit comparative assessments in reward circuit function in inbred and mutant mouse strains.

neuroscience↗

bioRxiv2020

Divergent behavioral consequences of manipulations enhancing pyramidal neuron excitability in the prelimbic cortex

BackgroundDrug-induced neuroadaptations in the prefrontal cortex are thought to underlie impaired executive functions that reinforce addictive behaviors. Repeated cocaine exposure increased layer 5/6 pyramidal neuron excitability in the mouse prelimbic cortex (PL), an adaptation attributable to a suppression of G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel activity. GIRK channel suppression in the PL of drug-naive mice enhanced the motor-stimulatory effect of cocaine. The impact of cocaine on PL GABA neurons, key pyramidal neuron regulators, and the behavioral relevance of increased PL pyramidal neuron excitability, remain unclear. MethodsThe effect of repeated cocaine on mouse layer 5/6 PL GABA neurons was assessed using slice electrophysiology. Adaptations enhancing PL pyramidal neuron excitability were modeled in drug-naive mice using persistent viral Cre ablation and acute chemogenetic approaches. The impact of these manipulations on PL-dependent behavior was assessed in motor activity and trace fear conditioning tests. ResultsRepeated cocaine treatment did not impact GIRK channel activity in, or excitability of, layer 5/6 PL GABA neurons. GIRK channel ablation in PL pyramidal neurons enhanced the motor-stimulatory effect of cocaine but did not impact baseline activity or fear learning. In contrast, direct or indirect chemogenetic activation of PL pyramidal neurons increased baseline and cocaine-induced motor activity and disrupted fear learning. These effects were mirrored by chemogenetic activation of PL pyramidal neurons projecting to the ventral tegmental area. ConclusionsManipulations enhancing the excitability of PL pyramidal neurons, including those projecting to the VTA, recapitulate behavioral hallmarks of repeated cocaine exposure.

neuroscience↗

bioRxiv2020

Bidirectional sex-dependent regulation of α6 and β3 nicotinic acetylcholine receptors by protein kinase Cε

Nicotine and alcohol are the most commonly abused substances worldwide, and comorbid nicotine and alcohol addiction is highly prevalent. Nicotinic acetylcholine receptors (nAChRs) containing the 6 and {beta}3 subunits are expressed in neural reward circuits and are critical for both nicotine and alcohol reward. nAChRs are dynamically regulated by signaling molecules such as protein kinase C epsilon (PKC{varepsilon}), which impact transcription of 6 and {beta}3 subunit mRNA (Chrna6 and Chrnb3, respectively). Previous work found decreased expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain of male PKC{varepsilon}-/- mice, who also consume less nicotine and alcohol compared to wild-type (WT) littermates. Here, we show that female PKC{varepsilon}-/- mice have enhanced expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain, which functionally impacts nAChR-dependent behavior, as female but not male PKC{varepsilon}-/- mice exhibit locomotor hypersensitivity to nicotine. Female PKC{varepsilon}-/- mice show no differences in alcohol-induced sedation compared to WT littermates, while male PKC{varepsilon}-/- have enhanced sedation compared to WT mice, a phenotype that has previously been reported in 6-/- mice. Female PKC{varepsilon}-/- mice also show reduced depression-like behavior in response to systemic injections of varenicline compared to WT littermates, and this effect was absent in male mice. Additionally, we found that female PKC{varepsilon}-/- mice show altered alcohol and nicotine consumption patterns in chronic voluntary two bottle choice assays. Our data reveal a bidirectional effect of sex in the transcriptional regulation of nicotinic receptors by PKC{varepsilon}, and highlight the importance of studying both sexes in preclinical animal models.

neuroscience↗

bioRxiv2019

Unequal interactions between alcohol and nicotine co-consumption: Suppression and enhancement of concurrent drug intake

RationaleAlcohol and nicotine addiction are prevalent conditions that co-occur. Despite the prevalence of co-use, factors that influence the suppression and enhancement of concurrent alcohol and nicotine intake are largely unknown.\n\nObjectivesOur goals were to assess how nicotine abstinence and availability influenced concurrent alcohol consumption, and to determine the impact of quinine adulteration of alcohol on aversion resistant alcohol consumption and concurrent nicotine consumption.\n\nMethodsMale and female C57BL/6J mice voluntarily consumed unsweetened alcohol, nicotine and water in a chronic 3-bottle choice procedure. In Experiment 1, nicotine access was removed for 1 week and re-introduced the following week, while the alcohol and water bottles remained available at all times. In Experiment 2, quinine (100-1000 M) was added to the 20% alcohol bottle, while the nicotine and water bottles remained unaltered.\n\nResultsIn Experiment 1, we found that alcohol consumption and preference were unaffected by the presence or absence of nicotine access in both male and female mice. In Experiment 2a, we found that quinine temporarily suppressed alcohol intake and enhanced concurrent nicotine, but not water, preference in both male and female mice. In Experiment 2b, chronic quinine suppression of alcohol intake increased nicotine consumption and preference in female mice without affecting water preference, whereas it increased water and nicotine preference in male mice.\n\nConclusionsQuinine suppression of alcohol consumption enhanced the preference for concurrent nicotine preference in male and female mice, suggesting that mice compensate for the quinine adulteration of alcohol by increasing their nicotine preference.

pharmacology and toxicology↗