Intra-carotid hyperosmotic stimulation increases Fos staining in forebrain organum vasculosum laminae terminalis neurones that project to the hypothalamic paraventricular nucleus

Abstract

Body fluid hyperosmolality has long been known to elicit homeostatic responses that range from drinking to inhibition of salt appetite to release of neurohypohyseal hormones (i.e. vasopressin and oxytocin). More recently, it has been recognized that hyperosmolality is capable of also provoking a significant increase of sympathetic nerve activity (SNA). It has been reported that neurones in the forebrain organum vasculosum laminae terminalis (OVLT) and hypothalamic paraventricular nucleus (PVN) each contribute significantly to this response. Here we sought to determine if sympathoexcitatory levels of hyperosmolality activate specifically those OVLT neurones that form a monosynaptic pathway to the PVN. First, we established in anaesthetized rats that graded concentrations of hypertonic NaCl (1.5 and 3.0 osmol kg(-1)) elicit graded increases of renal SNA (RSNA) when infused at a rate of 0.1 ml min(-1) through an internal carotid artery (ICA) - the major vascular supply of the forebrain. Next, infusions were performed in conscious rats in which OVLT neurones projecting to the PVN (OVLT-PVN) were retrogradely labelled with cholera toxin subunit B (CTB). Immunostaining of the immediate early gene product Fos and CTB was performed to quantify osmotic activation of OVLT-PVN neurones. ICA infusions of hypertonic NaCl and mannitol each significantly (P < 0.01-0.001) increased the number of Fos immunoreactive (Fos-ir) neuronal nuclei in the dorsal cap (DC) and lateral margins (LM) of OVLT. In the LM, infusions of 1.5 and 3.0 osmol kg(-1) NaCl produced similar increases in the number of Fos-ir neurones. In the DC, these infusions produced graded increases in Fos expression. Among OVLT neurones with axons projecting directly to the PVN (i.e. CTB-ir), graded hypertonic NaCl infusions again produced graded increases in Fos expression and this was observed in both the DC and LM. Although the DC and LM contained a similar number of OVLT-PVN neurones, the proportion of such neurones that expressed Fos-ir in responses to ICA hypertonic NaCl infusions was greater in the DC (P < 0.001). These findings support the conclusion that PVN-projecting neurones in the DC and LM of OVLT could participate in behavioural, neuroendocrine, and sympathetic nervous system responses to body fluid hyperosmolality.

Figure 1. Effect of ICA infusions of NaCl on RSNA and ABP in anaesthetized rats

Infusions (0.1 ml min⁻¹, 10 min) of 0.3 osmol kg⁻¹ (A), 1.5 osmol kg⁻¹ (B) and 3.0 osmol kg⁻¹ (C) NaCl via an internal carotid artery (ICA) produced graded increases of renal sympathetic nerve activity (RSNA) and arterial blood pressure (ABP) in anaesthetized rats. Summary data are provided in the Results. ∫RSNA, full-wave rectified and integrated (time constant = 3 s) RSNA.

Figure 2. Subdivisions of OVLT used for quantifying Fos- and CTB-positive neurones

Photomicrograph of an OVLT tissue section (40 μm) stained with neutral red. Shaded areas indicate the dorsal cap (DC) and lateral margins (LM) of OVLT where Fos-ir nuclei and CTB-ir neurones were counted for comparison across groups of rats receiving infusions of NaCl, mannitol, and phenylephrine. Additional details are provided in Methods.

Figure 3. Retrograde labelling of OVLT neurones projecting to the hypothalamic PVN

A, the retrograde tracer cholera toxin subunit B (CTB, green) was delivered bilaterally into the PVN by iontophoresis. Depositions of CTB were confined within the PVN and largely covered the rostro-caudal extent of the nucleus. B, CTB-ir neuronal somata were present in both the dorsal cap (DC) and lateral margins (LM) of OVLT. C, the number of CTB-ir OVLT neurones in the DC and LM did not differ across treatment groups.

Figure 4. Induction of Fos expression in OVLT by ICA hyperosmolality

ICA infusion of isotonic (0.3 osmol kg⁻¹) NaCl (A) and i.v. infusions of hypertonic (3.0 osmol kg⁻¹) NaCl (F) did not increase Fos-ir nuclei in OVLT compared to untreated rats (D). ICA infusions of 1.5 (B) and 3.0 osmol kg⁻¹ (C) hypertonic NaCl produced corresponding increases in Fos staining. Induction of Fos by ICA infusion of equal osmolal concentrations (1.5 osmol kg⁻¹) of NaCl (B) and mannitol (E) produced similar increases in Fos in the DC and LM. Consistent with hyperosmolality having a central action, Fos induction by 3.0 osmol kg⁻¹ hypertonic NaCl was greater when delivered by ICA (C) than i.v. (F) infusion. Fos induced by ICA NaCl was not likely to be an indirect effect of the accompanying pressor response since ICA and i.v. infusions produced similar average and peak increases of MAP (G, top vs. middle). Moreover, i.v. infusion of phenylephrine to mimic the amplitude and duration of the pressor response to ICA 3.0 osmol kg⁻¹ NaCl (G, top vs. bottom) revealed that the haemodynamic response by itself did not induce Fos (see H). Summary data of Fos staining in the DC and LM of OVLT (H) revealed that ICA hypertonic NaCl induced graded increases in Fos in the DC. In contrast, Fos staining in the LM appeared to saturate at the lower concentration (1.5 osmol kg⁻¹) of hypertonic NaCl. See Discussion for additional details. Scale bars in A–F= 100 μm. *P < 0.01 vs. ICA isotonic (0.3 osmol kg⁻¹) NaCl value in corresponding region. †P < 0.001 vs. 1.5 osmol kg⁻¹ NaCl value in corresponding region. §P < 0.001 vs. 1.5 osmol kg⁻¹ NaCl in DC.

Figure 5. Induction of Fos expression in OVLT-PVN neurones by ICA hyperosmolality

CTB- and Fos-positive neurones were numerous in both the DC and LM of OVLT (A). CTB-ir (PVN projecting) neurones that also expressed Fos in response to ICA hyperosmolality were likewise observed in both the DC (B) and LM (C) of OVLT. Summary data (D) indicate that the number of neurones double-labelled with Fos + CTB was not different in untreated rats, rats receiving ICA isotonic (0.3 osmol kg⁻¹) NaCl or i.v. hypertonic (3.0 osmol kg⁻¹) NaCl. In contrast, ICA infusions of graded NaCl concentrations caused the number of neurones double-labelled with Fos + CTB (OVLT-PVN neurones) to increase in a graded fashion. ICA infusions of equivalent osmolal concentrations of NaCl and mannitol (1.5 osmol kg⁻¹) produced similar increases in the number of neurones positive for CTB + Fos, with a significantly greater number of double-labelled cells in the DC than LM (P < 0.01). *P < 0.01 vs. isotonic (0.3 osmol kg⁻¹) NaCl value in corresponding region. †P < 0.001 vs. 1.5 osmol kg⁻¹ NaCl value in corresponding region. §P < 0.001 Vs. corresponding value in LM. Arrows in B and C indicate DC and LM neurones that colocalize Fos and CTB immunoreactivity.