Manuel Ferreira

Glucose effects on gastric motility and tone evoked from the rat dorsal vagal complex

1 To examine the effects of glucose on the central components of the vago‐vagal reflex control of gastric function, we performed both in vivo and in vitro experiments on neurones in the medial nucleus of the tractus solitarius (mNTS) and in the dorsal motor nucleus of the vagus (DMV). 2 In the in vivo anaesthetized rat preparation, unilateral microinjection of d‐glucose (10 or 50 mm (60 nl)−1) in mNTS produced inhibition of gastric motility and an increase in intragastric pressure. d‐glucose had no effect in the DMV. 3 In the in vitro rat brainstem slice preparation, whole‐cell recordings of DMV neurones showed that increasing the glucose concentration of the perfusion solution from 5 mm to 15 or 30 mm produced outward currents of 35 ± 5 pA (n= 7) and 51 ± 10 pA (n= 11), respectively. These were blocked by tetrodotoxin and picrotoxin, indicating that glucose was acting indirectly to cause the release of GABA. Decreasing the glucose concentration of the perfusing solution by one‐half produced an inward current of 36 ± 5 pA (n= 7). 4 Stimulation of the NTS evoked inhibitory postsynaptic currents (IPSCs) in DMV neurones. The amplitude of the evoked IPSCs was positively correlated with glucose concentration. Perfusion with the ATP‐sensitive K+ (KATP) channel opener diazoxide mimicked the effect of reduced glucose, while perfusion with the KATP channel blocker glibenclamide mimicked the effects of increased glucose. 5 Our data indicate that glucose had no direct excitatory effect on DMV neurones, but DMV neurones appear to be affected by an action of glucose on cell bodies of mNTS neurones via effects on an ATP‐sensitive potassium channel.

Axotomy and nerve growth factor regulate levels of neuronal nicotinic acetylcholine receptor α3 subunit protein in the rat superior cervical ganglion

Neuronal nicotinic acetylcholine receptors (nAChRs) play a significant role in sympathetic transmission in the superior cervical ganglia (SCG), with most of the signal carried by a nAChR containing an α3 subunit. Work has shown that transection of the postganglionic nerves (axotomy) of the SCG results in a decrease in mRNA transcripts for α3, α5, α7 and β4 and in protein expression of α7 and β4. To evaluate effects of axotomy on α3 protein in the SCG, quantitative immunoblotting was used to demonstrate a dramatic decrease (> 80%) in the levels of this subunit 4 days after axotomy. Similarly, immunocytochemistry showed a marked decline in the number and the intensity of stained neurons for the α3 subunit as well as tyrosine hydroxylase. Ganglia explanted into culture for 4 days also showed a substantial decrease in α3 subunit protein. This decrease was partially prevented by the addition of nerve growth factor (NGF) to the culture medium at the time of explantation. Additionally, this decrease was reversed by the addition of NGF to the culture medium following 4 days in culture in the absence of NGF. These findings suggest that the loss of α3 subunit contributes to the reported decrease in ganglionic synaptic transmission that follows axotomy, and that NGF plays an important role in regulating the expression of α3‐containing nAChRs in the SCG.

Nicotinic ACh receptor subtypes on gastrointestinally projecting neurones in the dorsal motor vagal nucleus of the rat

To determine the predominant nicotinic ACh receptor (nAChR) located on neurones in the dorsal motor nucleus of the vagus (DMV) that project to the gastrointestinal tract, we used the rat brainstem slice preparation and whole‐cell recordings of DMV neurones identified by retrograde DiI tracing to pharmacologically characterize nAChRs. Pressure ejection of acetylcholine (ACh, 250 μm for 200 ms) from a patch pipette placed ≈10‐20 μm from the surface of the recorded cell produced an inward current in most DMV neurones sampled. The average currents for neurones projecting to the fundus, antrum and caecum were 149 ± 38 (n= 25), 115 ± 18 (n= 29) and 117 ± 23 pA (n= 6), respectively. Blockade of the α7 subtype of nAChR with either α‐bungarotoxin (α‐BGT) or methyllycaconitine (MLA) counteracted 60‐75 % of the ACh‐evoked current in DMV neurones projecting to the fundus, antrum and caecum. In neurones projecting to the fundus and the antrum, currents resistant to α‐BGT were significantly blocked by dihydro‐β‐erythroidine (10‐20 nm), an antagonist of the α4β2 subtype of nAChR. In neurones projecting to the caecum, currents resistant to α‐BGT were significantly depressed by a low concentration of mecamylamine (1 μm). Cytisine (100 μm), an agonist of nAChRs that contain the α7 or the β4 subunit, evoked significant currents in caecum‐projecting neurones that were previously exposed to α‐BGT. In contrast, cytisine had no effect on DMV neurones previously exposed to α‐BGT that project to the fundus or antrum. Our data indicate that the prevailing nAChR subtype in DMV neurones projecting to the GI tract is the α7 subtype. In addition, we obtained evidence for the co‐expression of the α4β2 nAChR subtype on DMV neurones projecting to the fundus and antrum, and the α3β4 nAChR subtype on DMV neurones projecting to the caecum.