TH-Cre Mice Research Articles

1535-P: Physiological Role of Stress-Activated Neurons in the Paraventricular Hypothalamus

Psychological stimuli that disrupt homeostasis - stressors - lead to acute and chronic metabolic disturbance but the underlying neural circuits are incompletely understood. The paraventricular nucleus of the hypothalamus (PVH) plays a significant role in behavioral, autonomic and hormonal responses to stress. The PVH contains multiple cell types including neurons expressing tyrosine hydroxylase (TH). However, the contributions of PVHTH neurons to the acute stress response are largely unknown. We aim to determine the roles of PVHTH neurons in the metabolic and behavioral responses to acute stress. We first generated mice with tdTtomato expression in TH+ neurons (TH-tdTomato) by crossing validated TH-cre mice to mice with cre-dependent expression of tdTomato. Using immunohistochemistry for PVH neuropeptides linked to energy metabolism and stress in TH-Tdtomato mice, we quantified overlap of PVHTH neurons with CRH, oxytocin and vasopressin. We found small fractions of PVHTH neurons co-express CRH, oxytocin and vasopressin suggesting PVHTH neurons are a distinct population. Next, we assessed activation of PVHTH in response to acute stress by examining Fos expression in TH-tdTomato mice. Acute stress significantly increased the number of FOS+ PVHTH neurons. Finally, we examined whether chemogenetic activation of PVHTH neurons mimicked behavioral, hormonal and metabolic adaptations to stress using PVH injection of AAV with cre-dependent hM3DGq-mCherry or mCherry (control) in TH-cre mice. Chemogenetic activation of PVHTH neurons acutely suppressed dark-phase food intake and impaired glucose tolerance without effects on plasma corticosterone or anxiety-like behavior. These findings suggest acute stress may recruit PVHTH neurons to elicit hypophagia and impair glucose tolerance, independent of HPA axis activation and anxiety-like behavior. Future studies using chemogenetic inhibition will determine if PVHTH neurons are required for the full metabolic response to acute stress. Disclosure D.G.Espinoza: None. A.Alvarsson: None. M.Jimenez gonzalez: None. R.F.Hampton: None. R.Y.Li: None. K.Devarakonda: None. J.Choudhury: None. T.S.Waxman: None. S.Stanley: Consultant; Redpin Therapeutics.

Photostimulation of Ventral Tegmental Area-Insular Cortex Dopaminergic Inputs Enhances the Salience to Consolidate Aversive Taste Recognition Memory via D1-Like Receptors

Taste memory involves storing information through plasticity changes in the neural network of taste, including the insular cortex (IC) and ventral tegmental area (VTA), a critical provider of dopamine. Although a VTA-IC dopaminergic pathway has been demonstrated, its role to consolidate taste recognition memory remains poorly understood. We found that photostimulation of dopaminergic neurons in the VTA or VTA-IC dopaminergic terminals of TH-Cre mice improves the salience to consolidate a subthreshold novel taste stimulus regardless of its hedonic value, without altering their taste palatability. Importantly, the inhibition of the D1-like receptor into the IC impairs the salience to facilitate consolidation of an aversive taste recognition memory. Finally, our results showed that VTA photostimulation improves the salience to consolidate a conditioned taste aversion memory through the D1-like receptor into the IC. It is concluded that the dopamine activity from the VTA into IC is required to increase the salience enabling the consolidation of a taste recognition memory. Notably, the D1-like receptor activity into the IC is required to consolidate both innate and learned aversive taste memories but not appetitive taste memory.

Pituitary Adenylate Cyclase Activating Polypeptide Inhibits A10 Dopamine Neurons and Suppresses the Binge-like Consumption of Palatable Food

Pituitary adenylate cyclase-activating polypeptide (PACAP) binds to PACAP-specific (PAC1) receptors in multiple hypothalamic areas, especially those regulating energy balance. PACAP neurons in the ventromedial nucleus (VMN) exert anorexigenic effects within the homeostatic energy balance circuitry. Since PACAP can also reduce the consumption of palatable food, we tested the hypothesis that VMN PACAP neurons project to the ventral tegmental area (VTA) to inhibit A10 dopamine neurons via PAC1 receptors and KATP channels, and thereby suppress binge-like consumption. We performed electrophysiological recordings in mesencephalic slices from male PACAP-Cre and tyrosine hydroxylase (TH)-Cre mice. Initially, we injected PACAP (30 pmol) into the VTA, where it suppressed binge intake in wildtype male but not female mice. Subsequent tract tracing studies uncovered projections of VMN PACAP neurons to the VTA. Optogenetic stimulation of VMN PACAP neurons in voltage clamp induced an outward current and increase in conductance in VTA neurons, and a hyperpolarization and decrease in firing in current clamp. These effects were markedly attenuated by the KATP channel blocker tolbutamide (100 μM) and PAC1 receptor antagonist PACAP6–38 (200 nM). In recordings from A10 dopamine neurons in TH-Cre mice, we replicated the outward current by perfusing PACAP1–38 (100 nM). This response was again completely blocked by tolbutamide and PACAP6–38, and associated with a hyperpolarization and decrease in firing. These findings demonstrate that PACAP activates PAC1 receptors and KATP channels to inhibit A10 dopamine neurons and sex-dependently suppress binge-like consumption. Accordingly, they advance our understanding of how PACAP regulates energy homeostasis via the hedonic energy balance circuitry.

The Rigor and Transparency Index Quality Metric for Assessing Biological and Medical Science Methods.

SummaryThe reproducibility crisis is a multifaceted problem involving ingrained practices within the scientific community. Fortunately, some causes are addressed by the author's adherence to rigor and reproducibility criteria, implemented via checklists at various journals. We developed an automated tool (SciScore) that evaluates research articles based on their adherence to key rigor criteria, including NIH criteria and RRIDs, at an unprecedented scale. We show that despite steady improvements, less than half of the scoring criteria, such as blinding or power analysis, are routinely addressed by authors; digging deeper, we examined the influence of specific checklists on average scores. The average score for a journal in a given year was named the Rigor and Transparency Index (RTI), a new journal quality metric. We compared the RTI with the Journal Impact Factor and found there was no correlation. The RTI can potentially serve as a proxy for methodological quality.

Postganglionic sympathetic neurons, but not locus coeruleus optostimulation, activates neuromuscular transmission in the adult mouse in vivo

Recent work demonstrated that sympathetic neurons innervate the skeletal muscle near the neuromuscular junction (NMJ), and muscle sympathectomy and sympathomimetic agents strongly influence motoneuron synaptic vesicle release ex vivo. Moreover, reports attest that the pontine nucleus locus coeruleus (LC) projects to preganglionic sympathetic neurons and regulates human mobility and skeletal muscle physiology. Thus, we hypothesized that peripheral and central sympathetic neurons projecting directly or indirectly to the skeletal muscle regulate NMJ transmission. The aim of this study was to define the specific neuronal groups in the peripheral and central nervous systems that account for such regulation in adult mice in vivo by using optogenetics and NMJ transmission recordings in 3–5-month-old, male and female ChR2(H134R/EYFP)/TH-Cre mice. After detecting ChR2(H134R)/EYFP fluorescence in the paravertebral ganglia and LC neurons, we tested whether optostimulating the plantar nerve near the lumbricalis muscle or LC neurons effectively modulates motor nerve terminal synaptic vesicle release in living mice. Nerve optostimulation increased motor synaptic vesicle release in vitro and in vivo, while the presynaptic adrenoceptor blockers propranolol (β1/β2) and atenolol (β1) prevented this outcome. The effect is primarily presynaptic since miniature end-plate potential (MEPP) kinetics remained statistically unmodified after stimulation. In contrast, optostimulation of LC neurons did not regulate NMJ transmission. In summary, we conclude that postganglionic sympathetic neurons, but not LC neurons, increased NMJ transmission by acting on presynaptic β1-adrenergic receptors in vivo.

Altered baseline and amphetamine-mediated behavioral profiles in dopamine transporter Cre (DAT-Ires-Cre) mice compared to tyrosine hydroxylase Cre (TH-Cre) mice.

Transgenic mouse lines expressing Cre-recombinase under the regulation of either dopamine transporter (DAT) or tyrosine hydroxylase (TH) promoters are commonly used to study the dopamine (DA) system. While use of the TH promoter appears to have less liability to changes in native gene expression, transgene insertion in the DAT locus results in reduced DAT expression and function. This confound is sometimes overlooked in genetically targeted behavioral experiments. We sought to evaluate the suitability of DAT-Ires-Cre and TH-Cre transgenic lines for behavioral pharmacology experiments with DA agonists. We hypothesized that DAT-Ires-Cre expression would impact DAT-mediated behaviors, but no impact of TH-Cre expression would be observed. DAT-Ires-Cre and TH-Cre mice bred on mixed 129S6/C57BL/6 and pure C57BL/6 backgrounds were evaluated for novelty-induced, baseline, and amphetamine (AMPH)-induced locomotion, and for AMPH and D1 agonist (SKF-38393)-induced preservative behaviors. DAT-Ires-Cre mice on both mixed 129S6/C57BL/6 and pure C57BL/6 backgrounds displayed increased novelty-induced activity and decreased AMPH-induced locomotion, with mixed results for AMPH-induced stereotypy. TH-Cre mice on both backgrounds showed typical baseline activity and AMPH-induced stereotypy, with a difference in AMPH-induced locomotion observed only on the mixed background. Both transgenic lines displayed unaltered SKF-38393-induced grooming behavior. Our findings indicate that the DAT-Ires-Cre transgenic line may lead to confounds for experiments that are dependent on DAT expression. The TH-Cre transgenic line studied here may be a more useful option, depending on background strain, because of its lack of baseline and drug-induced phenotypes. These data highlight the importance of appropriate controls in studies employing transgenic mice.

Loss of striatal tyrosine-hydroxylase interneurons impairs instrumental goal-directed behavior.

The striatum mediates a broad range of cognitive and motor functions. Within the striatum, recently discovered tyrosine hydroxylase expressing interneurons (THINs) provide a source of intrastriatal synaptic connectivity that is critical for regulating striatal activity, yet the role of THIN's in behavior remains unknown. Given the important role of the striatum in reward-based behaviors, we investigated whether loss of striatal THINs would impact instrumental behavior in mice. We selectively ablated striatal THINs in TH-Cre mice using chemogenetic techniques, and then tested THIN-lesioned or control mice on three reward-based striatal-dependent instrumental tests: (a) progressive ratio test; (b) choice test following selective-satiety induced outcome devaluation; (c) outcome reinstatement test. Both striatal-THIN-lesioned and control mice acquired an instrumental response for flavored food pellets, and their behavior did not differ in the progressive ratio test, suggesting intact effort to obtain rewards. However, striatal THIN lesions markedly impaired choice performance following selective-satiety induced outcome devaluation. Unlike control mice, THIN-lesioned mice did not adjust their choice of actions following a change in outcome value. In the outcome reinstatement test THIN-lesioned and control mice showed response invigoration by outcome presentation, suggesting the incentive properties of outcomes were not disrupted by THIN lesions. Overall, we found that striatal THIN lesions selectively impaired goal-directed behavior, while preserving motoric and appetitive behaviors. These findings are the first to describe a function of striatal THINs in reward-based behavior, and further illustrate the important role for intrastriatal interneuronal connectivity in behavioral functions ascribed to the striatum more generally.

Preserved dopaminergic homeostasis and dopamine-related behaviour in hemizygous TH-Cre mice.

Cre-driver mouse lines have been extensively used as genetic tools to target and manipulate genetically defined neuronal populations by expression of Cre recombinase under selected gene promoters. This approach has greatly advanced neuroscience but interpretations are hampered by the fact that most Cre-driver lines have not been thoroughly characterized. Thus, a phenotypic characterization is of major importance to reveal potential aberrant phenotypes prior to implementation and usage to selectively inactivate or induce transgene expression. Here, we present a biochemical and behavioural assessment of the dopaminergic system in hemizygous tyrosine hydroxylase (TH)-Cre mice in comparison to wild-type (WT) controls. Our data show that TH-Cre mice display preserved dopaminergic homeostasis with unaltered levels of TH and dopamine as well as unaffected dopamine turnover in striatum. TH-Cre mice also show preserved dopamine transporter expression and function supporting sustained dopaminergic transmission. In addition, TH-Cre mice demonstrate normal responses in basic behavioural paradigms related to dopaminergic signalling including locomotor activity, reward preference and anxiolytic behaviour. Our results suggest that TH-Cre mice represent a valid tool to study the dopamine system, though careful characterization must always be performed to prevent false interpretations following Cre-dependent transgene expression and manipulation of selected neuronal pathways.

EP 56. Optogenetic control of the locus coeruleus in mice: Behavioral and electrophysiological data

The locus coeruleus (LC) is the sole source of noradrenergic projections to the cortex and to better understand its essential role for attention-dependent cognitive processes, we first expressed the halorhodopsin eNpHR3.0 to reversibly silence LC activity in naive male B6.Cg-Tg(Th-cre)1Tmd/J hemizygous mice (Th-cre mice) that express Cre-recombinase under the control of the tyrosine hydroxylase (TH) promoter. Unilateral optogenetic silencing of the LC during the performance of an attentional set-shifting task (ASST) selectively impaired learning of those parts of the ASST that most strongly rely on cognitive flexibility. In particular, extra-dimensional set-shifting (EDS) and reversal learning was impaired. However, those parts of the task that are less dependent on cognitive flexibility, i.e. compound discrimination (CD) and the intra-dimensional shifts (IDS), as well as attentional set formation were unaffected by LC silencing. Therefore, our results suggest a modulatory influence of the LC on cognitive flexibility, mediated by different frontal networks. Subsequently, we used AAV8-Ef1a-DIO-Chronos-eYFP to stimulate LC activity in Th-cre mice and performed electrophysiological recordings during optogenetic stimulation by using an optetrode. Laser pulses of different intensities (0.5, 1, 2, 5, and 10 mW) and different pulse durations (5, 10, 20, 50, 100, 200, and 500 ms) were tested. Results showed that pulses at 5 mW of 5 ms pulse duration are optimal and that those laser pulses are able to induce bursts of 20 Hz discharges and sustained 10 Hz discharges of LC neurons.

Exposure to cocaine regulates inhibitory synaptic transmission from the ventral tegmental area to the nucleus accumbens

Synaptic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) make up the backbone of the brain reward pathway, a neural circuit that mediates behavioural responses elicited by natural rewards as well as by cocaine and other drugs of abuse. In addition to the well-known modulatory dopaminergic projection, the VTA also provides fast excitatory and inhibitory synaptic input to the NAc, directly regulating NAc medium spiny neurons (MSNs). However, the cellular nature of VTA-to-NAc fast synaptic transmission and its roles in drug-induced adaptations are not well understood. Using viral-mediated in vivo expression of channelrhodopsin 2, the present study dissected fast excitatory and inhibitory synaptic transmission from the VTA to NAc MSNs in rats. Our results suggest that, following repeated exposure to cocaine (15 mg kg(-1) day(-1) × 5 days, i.p., 1 or 21 day withdrawal), a presynaptic enhancement of excitatory transmission and suppression of inhibitory transmission occurred at different withdrawal time points at VTA-to-NAc core synapses. In contrast, no postsynaptic alterations were detected at either type of synapse. These results suggest that changes in VTA-to-NAc fast excitatory and inhibitory synaptic transmissions may contribute to cocaine-induced alteration of the brain reward circuitry.

Bone Marrow Neuropathy Prevents Hematopoietic Regeneration

Abstract 139Chemotherapy regimens commonly produce long-term hematopoietic injury, possibly through a lesion to the hematopoietic stem cell (HSC) and/or its niche but the mechanisms have yet to be elucidated. Nestin-expressing mesenchymal stem cells, under regulation of the sympathetic nervous system (SNS), are a critical component of the HSC niche (Nature. 2010; 466:829). Since peripheral neuropathy is a common complication of chemotherapy, we hypothesized that chemotherapy-induced sympathetic bone marrow (BM) neuropathy could impair BM regeneration. To test this issue, we sympathectomized mice using 6-hydroxydopamine (6OHDA) or diphtheria toxin (DT) injection into TH-Cre-iDTR mice, which express the DT receptor exclusively on catecholaminergic neurons. In both models, SNS ablation led to reduced BM regeneration following 5-fluorouracil (5FU) challenge, as determined by the delayed recovery in BM cellularity (1.7-fold↓*), clonogenic progenitors (CFU-C; 2.5-fold↓*) and Lin−Sca-1+c-Kit+Flt3− cells (LSKF cells 2.5-fold↓*). Further, inhibition of β3 adrenergic signaling with the antagonist SR5920A impaired BM recovery to the same extent as 6OHDA sympathectomy: BM cells (1.6-fold↓*), CFU-C (1.6-fold↓*), LSKF (1.6-fold↓*). We then treated mice with the chemotherapy drug cisplatin, which induces neuropathies in mice (2.8-fold↓* in SNS fibers in BM). Cisplatin-treated mice exhibited reduced survival after BM transplantation (66% vs 100%* for cisplatin-treated and control, respectively) due to delayed regeneration: BM cells (4.6-fold↓*), CFU-C (8.8-fold↓*), LSKF (6.4-fold↓*). To evaluate the potential benefit of neuroprotection, we treated mice with 4-methylcatechol (4-MC), a powerful inducer of Nerve Growth Factor (NGF), together with cisplatin or 6OHDA, which led to the protection of SNS fibers (1.8-fold↑* and 11-fold↑* respectively). This prevented transplantation-associated death and increased BM recovery following transplantation or 5FU administration: BM cells (1.9- and 1.6-fold↑, respectively *), CFU-C (3.6- and 1.4-fold↑*), LSKF (4.5- and 2.4-fold↑*). To ascertain whether 4-MC/NGF acted specifically on the nervous system, rather than on hematopoietic or stromal cells, we used TrkAneo/neo mice, which are genetically deficient in the NGF receptor in non-neuronal tissues (Development. 2004;131:5185). Administration of 4-MC to sympathectomized TrkAneo/neo mice enhanced BM regeneration after 5FU: BM cells (2.0-fold↑*), CFU-C (2.0-fold↑*), LSKF (2.2-fold↑*), suggesting that 4-MC acts directly on neurons to rescue hematopoiesis. Since deletion of p53 promotes neuron survival after genotoxic damage (Neurodegeneration. 1996;5:233), we intercrossed mice expressing TH-Cre mice with p53flox/flox animals to delete p53 specifically in catecholaminergic neurons. SNS fibers of TH-Cre;p53flox/flox mice were protected after cisplatin treatment compared to TH-Cre;p53+/flox controls, and this was associated with improved BM recovery after transplantation: BM cells (2.4-fold↑*), CFU-C (2.9-fold↑*), LSKF (6.5-fold↑*) when compared to TH-Cre;p53+/flox animals. Thus, these various pharmacologic and genetic models strongly suggest that chemotherapy-induced sympathetic neuropathy prevents BM regeneration. To evaluate further the mechanisms, we investigated whether BM neuropathy might lead to dysfunctional HSC niche. SNS injury using 6OHDA or cisplatin treatment led to Nestin+ cell expansion and accumulation (1.7- and 3.8-fold↑*, respectively). Treatment with 4-MC reverted this phenotype (1.8-fold↓* for 6OHDA and 1.5-fold↓* for cisplatin), indicating that Nestin+ cell expansion is due to reduced SNS signaling. SNS injury also led to increased Nestin+ cell sensitivity to genotoxic insult as determined by increased Nestin+ cell death and apoptosis (1.4-fold↑*) 24h after 5FU injection, which reduced Nestin+ cell numbers during regeneration (2.1-fold↓* 12 days after 5FU). By contrast, the number of osteoblasts and endothelial cells were unaffected. These results indicate that adrenergic signals promote the quiescence of HSC niche cells and that sympathetic neuropathy renders the niche vulnerable to genotoxic insult, impairing hematopoietic regeneration. Neural protection may represent a novel strategy to maintain hematopoietic reserve and enhance transplantation efficiency in chemotherapy-treated patients. *P<0.05 Disclosures:No relevant conflicts of interest to declare.

Semaphorin 3F is a bifunctional guidance cue for dopaminergic axons and controls their fasciculation, channeling, rostral growth, and intracortical targeting.

Dopaminergic neurons in the mesodiencephalon (mdDA neurons) make precise synaptic connections with targets in the forebrain via the mesostriatal, mesolimbic, and mesoprefrontal pathways. Because of the functional importance of these remarkably complex ascending axon pathways and their implication in human disease, the mechanisms underlying the development of these connections are of considerable interest. Despite extensive in vitro studies, the molecular determinants that ensure the perfect formation of these pathways in vivo remain mostly unknown. Here, we determine the embryonic origin and ontogeny of the mouse mesoprefrontal pathway and use these data to reveal an unexpected requirement for semaphorin 3F (Sema3F) and its receptor neuropilin-2 (Npn-2) during mdDA pathway development using tissue culture approaches and analysis of sema3F(-/-), npn-2(-/-), and npn-2(-/-);TH-Cre mice. We show that Sema3F is a bifunctional guidance cue for mdDA axons, some of which have the remarkable ability to regulate their responsiveness to Sema3F as they develop. During early developmental stages, Sema3F chemorepulsion controls previously uncharacterized aspects of mdDA pathway development through both Npn-2-dependent (axon fasciculation and channeling) and Npn-2-independent (rostral growth) mechanisms. Later on, chemoattraction mediated by Sema3F and Npn-2 is required to orient mdDA axon projections in the cortical plate of the medial prefrontal cortex. This latter finding demonstrates that regulation of axon orientation in the target field occurs by chemoattractive mechanisms, and this is likely to also apply to other neural systems. In all, this study provides a framework for additional dissection of the molecular basis of mdDA pathway development and disease.

Bcl-x is required for proper development of the mouse substantia nigra.

Recent findings have uncovered a role for the Bcl-x gene in the survival of dopaminergic neurons. The exact nature of this role has been difficult to examine because of the embryonic lethality of Bcl-x gene disruption in mouse models. Here we report the generation catecholaminergic cell-specific conditional Bcl-x gene knock-out mice using Cre-lox recombination technology. First we produced transgenic mice that express Cre recombinase from an exogenous rat tyrosine hydroxylase promoter (TH-Cre mice). These mice were crossed to Z/AP and Z/EG reporter mouse strains to verify catecholaminergic (TH-positive) cell-specific Cre expression. The TH-Cre mice then were mated to mice possessing the Bcl-x gene flanked by loxP sites, thereby producing offspring with Bcl-x deletion limited to catecholaminergic cells. The resulting mice are viable but have one-third fewer catecholaminergic neurons than do control animals. They demonstrate a deficiency in striatal dopamine and also tend to be smaller and have decreased brain mass when compared with controls. Surprisingly, surviving neurons were found that lacked Bcl-x immunoreactivity, thereby demonstrating that this gene is dispensable for the ongoing survival of a subpopulation of catecholaminergic cells.

Transgenic mice engineered to target Cre/loxP-mediated DNA recombination into catecholaminergic neurons.

To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We crossbred TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.