ABSTRACT
Aim To observe the effect of corticotropin-releasing factor ( CRF) -expressing neurons on presympathetic neurons in hypothalamic paraventricular nucleus ( PVN) of normotensive Wistar Kyoto ( WKY) rats or spontaneously hypertensive rats (SHR) , and to elucidate the underlying neuronal circuit mechanism of central sympathetic hyperexcitability. Methods The expression levels of CRF protein in WKY rats and SHR PVN were determined by Western blot. Meanwhile, the WKY and SHR PVN CRF-expressing neurons and presympathetic neurons were observed by immunofluo-rescent staining. Adult WKY rats and SHR were used in this study. By microinjection of Cre-dependent ade-no-associated viruses ( AAV) that specifically recognized the CRF promoter and AAV of chemogenetics into the PVN, CRF-expressing neurons expressed designer receptors exclusively activated by designer drugs (DREADDs). Human M3 muscarinic DREADD coupled to Gq receptor ( hM3 Dq) was specifically expressed in PVN CRF-expressing neurons in WKY rats, while human M4 muscarinic DREADD coupled to Gi receptor ( hM4Di) was specifically expressed in PVN CRF-expressing neurons in SHR. Clozapine-N-oxide (CNO) , as a designer ligand, would couple to excitatory hM3Dq or inhibitory hM4Di to regulate the excitability of PVN CRF-expressing neurons. Then the PVN presympathetic neurons were retrogradely labeled by microinjection of fluosecent tracer into the intermedio-lateral column (IML) of spinal cord. Lastly, whole cell patch clamp was used to determine the effect of CNO (10 jjumol L~ ) on spontaneous excitatory postsynaptic currents ( sEPSCs) and current-evoked firing of PVN presympathtic neurons of WKY rats and SHR. Results The expression of CRF protein in the PVN of SHR was significantly higher than that of WKY rats, and the activity and number of CRF-expressing neurons in the PVN of SHR were increased. PVN CRF-expressing neurons were expressed with chemogenetic DREADDs and PVN presympathetic neurons were retrogradely labeled with fluorescent tracer in WKY rats and SHR. In SHR expressed with chemogenetic inhibitory hM4Di-mCherry of PVN CRF-expressing neurons, bath application of CNO to the brain slices resulted in a significant decrease in sEPSCs frequency, but no change in their amplitude of labeled PVN presympathetic neurons. In contrast, in WKY rats expressed with excitatory hM3Dq-eGFP of PVN CRF-expressing neurons, CNO had no obvious effect on the sEPSCs frequency and amplitude in PVN presympathetic neurons. Furthermore, bath application of CNO had no significant effect on current-evoked firing of PVN presympathetic neurons of either WKY rats with hM3Dq-eGFP expression in CRF neurons or SHR with hM4Di-mCherry expression in CRF neurons. Conclusions The activity and number of PVN CRF-expressing neurons are increased in SHR, and CRF-expressing neurons enhance the excitability of presympathetic neurons, which acts as a regulatory neuronal microcircuit between CRF neurons and presympathetic neurons in the PVN.
ABSTRACT
OBJECTIVE: To determine the cardiovascular actions of salusin-α in the caudal ventrolateral medulla (CVLM) in anesthetized rats. METHODS: Sixty-one anesthetic male SD rats were employed in the present study. The dose-dependant effects of salusin-α (0.04-4 pmol) on blood pressure and heart rate in the CVLM were determined by unilateral microinjection of salusin-α or artificial cerebrospinal fluid (aCSF) into the CVLM in 25 rats. In the other 36 rats, a CSF, kynurenic acid (Kyn), atropine, hexametho-nium (Hex) in the CVLM or a CSF/muscimol in the RVLM were given in advance to determine the mechanism of the cardiovascular actions of intra-CVLM salusin-α. RESULTS: Unilateral microinjection of salusin-a into the CVLM produced a dose-dependent hypotension and bradycardia. Prior administration of Kyn (1 nmol) or Hex (120 pmol) into the CVLM did not alter the hypotension and bradycardia induced by intra-CVLM salusin-α (P>0.05). But prior administration of atropine into the CVLM or pretreatment with muscimol within RVLM almost completely abolished the hypotension and bradycardia evoked by intra-CVLM salusin-α (P<0.05). CONCLUSION: Microinjection of salusin-α into the CVLM produces significantly hypotension and bradycardia, which probably originates from suppressing the activities of presympathetic neurons in the RVLM.