Blood pressure regulation in Thra1+/m mice. The hypotension observed in the recently identified patient with a mutant thyroid hormone receptor α1 (TRα1) allele (14) prompted us to investigate blood pressure and associated serum parameters in our Thra1+/m animal model. Surprisingly, despite strongly reduced pulmonary angiotensin-converting enzyme (Ace) expression and lower serum angiotensin II levels in the mutant animals (Figure 1A), we found that blood pressure was similar to that in wild-type controls (Figure 1B). We thus tested whether the reduced Ace expression was an acute consequence of the defective TRα1 signaling by treating Thra1+/m mice with supraphysiological doses of triiodothyronine (T3), which reactivates signaling through the mutant receptor (11). The treatment increased pulmonary Ace expression, normalized serum angiotensin levels (Figure 1C), and restored the bradycardia in the mice (12). Determination of blood pressure in the T3-treated animals, which now had normalized heart rate and angiotensin II levels, revealed a 25% increase in systolic, diastolic, and mean arterial pressure (Figure 1D), suggesting an additional defect in the control of cardiovascular function in Thra1+/m mice. As the phenotype was only revealed in the T3-treated adult animal, we hypothesized that a developmental defect residing in the central nervous system caused this phenotype.

Figure 1 Regulation of blood pressure in Thra1+/m mice before and after treatment with T3. (A) mRNA expression of renal renin (Ren1), hepatic angiotensinogen (Agt), and pulmonary Ace as well as serum aldosterone (Aldost) and angiotensin II (Ang II) levels in wild-type and Thra1+/m mice. (B) Systolic, diastolic, and mean arterial blood pressure (MAP) in wild-type and Thra1+/m mice. (C) Heart rate in wild-type and Thra1+/m mice before and after T3 treatment as well as pulmonary Ace mRNA expression and serum angiotensin II levels in T3-treated animals. (D) Systolic, diastolic, and mean arterial blood pressure in T3-treated wild-type and Thra1+/m mice. All values are mean ± SEM; n = 5. **P < 0.01. NS, not significant.

Fewer pv neurons in the anterior hypothalamus of Thra1+/m mice. Our previous studies already indicated a defect in the central autonomic control of cardiovascular function (12). Thus, we examined the cellular composition of the hypothalamus — the master regulator of the autonomic nervous system (15). The data revealed that the levels of pv mRNA were halved in the mutant animals (Supplemental Figure 1A; supplemental material available online with this article; doi: 10.1172/JCI65252DS1). A subsequent immunohistochemical analysis identified an approximately 70% reduction of a previously unknown population of small hypothalamic pv+ cells (Figure 2, A and D) localized in the AHA (Supplemental Figure 1D). Cells expressing other calcium-binding marker proteins, such as calretinin and calbindin, were unaffected (Supplemental Figure 1, A and B) as well as a more distant nucleus of pv+ cells in the lateral hypothalamic area (Supplemental Figure 1B) described recently (16). Similar to pv+ neurons in the cortex (17), the AHA pv+ cells also appeared between postnatal days 7 and 14 (Supplemental Figure 1C).

Figure 2 Reduced number of pv cells in the anterior hypothalamus of Thra1+/m mice. (A) Immunohistochemistry for pv in the anterior hypothalamus, as overview (left; scale bar: 250 μm) and high magnification (right; scale bar: 50 μm) in wild-type and Thra1+/m mice (middle; scale bar: 250 μm). fx, fornix; mt, mamillothalamic tract; PVN, paraventricular nucleus of the hypothalamus; 3V, 3rd ventricle; opt, optic tract. (B) Double immunohistochemistry for GFP (green) and pv (red) in the AHA of a mouse strain expressing a chimeric TRα1-GFP protein. Yellow indicates overlapping staining. Scale bar: 25 mm. (C) pv neurons in T3-treated wild-type and Thra1+/m mice or crossings with hyperthyroid Thrb–/– mice. Scale bar: 250 μm. (D) Quantification of pv neurons in the AHA of the different animal models. All values are mean ± SEM; n = 4–9. *P < 0.05 to untreated wild type; ***P < 0.001 to untreated wild type; #P < 0.05 to untreated Thra1+/m.

As we detected the presence of TRα1 protein in the pv+ cells (Figure 2B), we tested whether a reactivation of the mutant receptor through increased thyroid hormone levels (11) would restore the number of cells in Thra1+/m mice — either by a 14-day oral thyroid hormone treatment or through genetic inactivation of thyroid hormone receptor β (TRβ) that causes hyperthyroidism in the animals throughout their postnatal life (18). Neither condition led to normalization; in contrast, the lack of TR further reduced the number of cells in Thra1+/mThrb–/– and Thra1+/+Thrb–/– animals (Figure 2, C and D). This observation demonstrates that intact thyroid hormone signalling via both TR isoforms is required for proper pv+ cell development in the AHA and that the cells are absent in Thra1+/m mice rather than exhibiting a diminished pv expression due to impaired TRα1 signaling.

AHA pv+ cells respond to temperature alterations and thyrotropin-releasing hormone stimulation. To obtain information on a possible function of the AHA pv+ neurons, we performed whole-cell patch-clamp recordings (Figure 3A) in hypothalamic slices of adult mice expressing GFP under the pv promoter (19). While most of the cells were not responsive to angiotensin II (Figure 3B), all tested AHA pv+ cells responded to alterations in temperature ranging from 25°C to 40°C. Sixty-nine percent of the cells were excited by increasing temperature with reversible depolarization and increase in action potential discharge, whereas 31% were inhibited and showed reversible hyperpolarization and cessation of action potential discharge (Figure 3C). This sensitivity persisted even after blocking synaptic transmission with tetrodotoxin, demonstrating that the thermosensitivity of pv+ AHA neurons is an intrinsic property and not the consequence of other neuronal inputs (Supplemental Figure 2, A and B). No temperature sensitivity was observed in pv+ neurons from the cortex in control experiments (Supplemental Figure 2B).

Figure 3 Electrophysiological responses of pv+ cells in the AHA. (A) Differential interference contrast (DIC) micrograph showing a recorded AHA pv+ neuron (indicated by an asterisk) (left; scale bar: 200 μm) and higher-magnification images of the same GFP-positive neuron under fluorescence and DIC (right; 500-fold magnification). (B) Response of AHA pv+ neurons to angiotensin II (82% no response; n = 14 out of 17). (C) Temperature responsiveness of the AHA pv+ cells to heat (31% inhibited, n = 5 out of 16, and 69% excited, n = 11 out of 16) in patch-clamp recordings on hypothalamic sections of transgenic pvGFP mice. (D) Response of AHA pv+ neurons to TRH (48% excited, n = 10 out of 21; 19% inhibited, n = 4 out of 21; and 33% nonresponsive, n = 7 out of 21) (the neuron in the top panel was held below threshold to prevent action potential firing; no holding current was applied in the other experiments).

To reveal the underlying molecular mechanism, we performed voltage clamp ramps in the heat-inhibited neurons. These experiments revealed a current reversal at –85 mV (Supplemental Figure 2, C and D), which — in conjunction with the depolarizing effects of the potassium channel blocker tolbutamide (Supplemental Figure 2B) — suggested an involvement of K+-ATP channel activation in the thermosensation of these cells. We also detected the temperature-activated transient receptor potential (TRP) channel TRPV4 on 56% of the AHA pv+ cells (Supplemental Figure 2E), which likely contributes to the depolarization observed in the heat-excited pv+ cells (20, 21). That also TRPM8 was detected on a majority of AHA pv+ cells (Supplemental Figure 2E), indicates a complex interplay between several thermosensitive channels (22).

To differentiate better between the cell types, we tested whether other substances would also elicit different electrophysiological responses specific to some AHA pv+ neurons. Thyrotropin-releasing hormone (TRH), known to have central effects on the control of the autonomic nervous system (23–25), excited 48% (Figure 3D) and inhibited 19% of the neurons. Some of these inhibitions were associated with an increase in inhibitory postsynaptic potentials (Supplemental Figure 2, F and G), suggesting that the AHA pv+ neurons are part of a TRH excited inhibitory network. However, there was no correlation between the TRH response of a neuron and the type of temperature sensitivity, suggesting that at least 4 different subpopulations exist among the AHA pv+ neurons.

Physiological role of the pv+ cells in the AHA. To understand the physiological function of the pv+ neurons, we aimed to ablate the cells in vivo by stereotaxic injection of a novel conditionally neurotoxic adeno-associated virus (AAV) into the AHA of pvCre transgenic mice (Figure 4A). As a result of diphtheria toxin A expression after Cre recombination, we achieved approximately 40% reduction of pv+ cells in the AHA of pvCre mice (Figure 4C, right). The presence of GFP-positive cells at the injection site (Figure 4B) demonstrated that the infection was not lethal for Cre-negative cells. This was further corroborated by the absence of alterations in other hypothalamic cell populations of AAV-injected pvCre mice (Supplemental Figure 1E), including the pv+ cells in the lateral hypothalamus (16). Moreover, the number of pv+ cells in the AHA of AAV-injected wild-type mice remained normal as expected (Figure 4C, left).

Figure 4 Effect of the in vivo ablation of AHA pv cells in pvCre mice. (A) AAV construct before and after Cre recombination. CMV, cytomegalovirus promotor; loxP, Cre recombination site; tpA, triple polyadenylation site; neoR, neomycin resistance gene; dtA, diphtheria toxin A. (B) Immunohistochemistry for EGFP at the site of the injection (indicated by asterisks) showing AAV-infected cells (scale bar: 250 μm). (C) Immunohistochemistry for pv in AAV-injected wild-type, nonablated pvCre, or AAV-injected ablated pvCre mice (the overall ablation efficiency is shown in the cell count at the bottom; ***P < 0.001 to nonablated, unpaired 2-tailed Student’s t test; the respective groups for the subsequent cardiac and metabolic analyses had cell counts of 81 ± 13 in the ablated animals vs. 142 ± 10 in the nonablated animals; n = 6, P = 0.002; scale bar: 500 μm). Asterisks indicate the site of injection. (D) Systolic, diastolic, and mean arterial blood pressure in mice with reduced numbers of pv+ cells in the AHA (black bars) and controls (white bars; *P < 0.05 for ablated vs. nonablated, unpaired 2-tailed Student’s t test). (E) Heart rates in these mice (*P < 0.05 for ablated vs. nonablated at 4°C, 2-way ANOVA). (F) Change in heart rate upon pharmacological deinnervation of the parasympathetic nervous system (PSNS) (scopolamine methyl bromide) or the sympathetic nervous system (SNS) (timolol) in mice with reduced numbers of pv+ cells in the AHA (black bars) and controls (white bars; *P < 0.05 for ablation, 2-way ANOVA). All values are mean ± SEM.

Subsequent physiological analyses after the virus-induced ablations revealed no immediate effects on body weight, food intake, overall activity, respiratory quotient, or body temperature when compared to those of nonablated pvCre controls (Supplemental Figure 3, A–G). Furthermore, no activation of the brown fat was detected upon ablation (Supplemental Figure 3, H and I); only a minor decrease in oxygen consumption and carbon dioxide production was observed at room temperature (Supplemental Figure 3, D and E). In contrast, we found a prominent hypertension in animals with AHA pv+ cell ablations (Figure 4D), with a 13% increase in systolic and a 22% increase in diastolic blood pressure. We did not observe any change in adrenal mRNA expression and aldosterone serum levels (Supplemental Figure 3J), serum angiotensin II levels (79% ± 18% of control levels, P = 0.98), or total T3 and thyroxine (T4) levels (total T3, 1.22 ± 0.18 nmol/l in control vs. 1.31 ± 0.28 nmol/l in ablated, P = 0.63; total T4, 46.25 ± 6.18 nmol/l in control vs. 46.20 ± 9.64 nmol/l in ablated, P = 0.99), suggesting that the AHA pv+ cells control cardiovascular function directly by the autonomic nervous system rather than through endocrine alterations.

To understand the cardiovascular phenotype better, radio telemetry transmitters implanted into the abdominal cavity were used to measure heart rate in conscious and unrestrained animals. This revealed a minor tachycardia at room temperature in the animals with ablated AHA pv+ cells, which was increased during night activity (Figure 4E and Supplemental Figure 3K). When exposed to cold, these mice exhibited a pronounced increase in heart rate, 24% higher than that in control animals (Figure 4E). Interestingly, the tachycardia disappeared entirely at thermoneutrality (Figure 4E).

Given the abnormal cardiovascular response to temperature and the fact that the autonomic innervation of the heart shifts in rodents with decreasing temperature (26), we hypothesized that the ablation of AHA pv+ cells changed the autonomic control of the cardiovascular system. To test this hypothesis, we performed a selective pharmacological deinnervation of the heart as described previously (12), using the muscarinic antagonist scopolamine methyl bromide and the β-adrenergic antagonist timolol (Supplemental Figure 3L). Indeed, the analysis revealed reduced sympathetic and parasympathetic input to the heart in mice with ablated AHA pv+ cells (Figure 4F), demonstrating the important role of these neurons in the autonomic control of cardiovascular function.