Prenatal X-irradiation increases GFAP- and calbindin D28k-immunoreactivity in the medial subdivision of the nucleus of solitary tract in the rat.

Glial fibrillary acidic protein- (GFAP) and calbindin D28k-immunoreactivity (IR) were investigated in the medial subdivision of the nucleus of the solitary tract (mNST) of prenatally X-irradiated rats. Pregnant rats were exposed to a single whole-body X-irradiation on day 11 or 16 of gestation at a dose of 1. 3 Gy. The offspring were killed at 7-14 days of age for the immunohistochemical observations. Rat pups showed strong GFAP-IR at the level rostral to the obex when receiving X-rays on day 11 of gestation, with hypertrophy of astrocyte cell bodies and cytoplasmic processes, but weak GFAP-IR when receiving X-rays on day 16 of gestation. Calbindin D28k-IR was stronger in the animals receiving X-rays on day 11 or 16 of gestation compared to that in the control animals. In the present study, the increase of GFAP- and calbindin D28k-IR cells in the mNST might indicate that adaptative mechanisms are taking place to preserve integrated nervous system function and possibly, to provide neuroprotection.

Synapse formation and spontaneous activity in rat brainstem neurons in primary culture.

The correlation between synaptogenesis and onset of spontaneous action potentials was assessed in rat brainstem cells up to 29 days in primary culture. Cells exhibited different stages of maturation followed by electron microscopy and patch clamp recordings. Terminal boutons with no preferential orientation of presynaptic vesicles appeared after 2 days in culture. After 5 days, preferential orientation of presynaptic vesicles and thickening of postsynaptic membranes were observed. The spontaneous discharge of action potentials, single or bursting, was observed after 7 days in vitro. This was followed by the expression of a 128-pS K(+) channel starting at 13 days in vitro. A 69-pS K(+) channel was also present throughout the duration of the cultures. These results suggest that spontaneous discharge of action potentials does not occur before synapses are formed and K(+) channel types develop differentially in brainstem neurons in vitro.

Ca2+ regulation of a large conductance K+ channel in cultured rat cerebellar Purkinje neurons.

The calcium sensitivity of a large conductance voltage-sensitive potassium channel found in cultured rat cerebellar Purkinje neurons was studied in membrane patches from the somatic region of the cultured Purkinje neurons using single-channel recording techniques. The potassium channel had a conductance of approximately 94 picosiemens (pS) under physiologic ionic conditions and was active at depolarized membrane potentials. Activity due to this channel type was not observed when the saline at the internal surface of the membrane was calcium free. Low intracellular calcium concentration (10 nM) triggered channel activity at depolarized membrane potentials. Channel activity increased further with increasing intracellular calcium concentrations and was evident at more negative membrane potentials. The high sensitivity of this potassium channel type to intracellular calcium and its abundance in the Purkinje neuron membrane may reflect a prominent role in the control of action potential duration and interspike interval when the neurons are firing in a rapid, repetitive mode.

Pontine and medullary control of the respiratory activity in the trigeminal and facial nerves of the newborn mouse: an in vitro study.

In vitro, the respiratory activity in rodents is characterized by: (i) the rapidly peaking, slowly decrementing pattern of spontaneous and rhythmic active phases recorded from the motor rootlets, and (ii) the specific location of their rhythmic generator in the ventrolateral medulla. The aim of the present study was to assess whether the trigeminal and facial motor rootlets still exhibit respiratory activity in the absence of peripheral and higher cerebral structures, and to compare the onset of their active phases with that of other respiratory rootlets, using the in vitro isolated brainstem--spinal cord preparation of the newborn mouse and rat. Spontaneous rhythmic activity was recorded from the trigeminal and facial rootlets. It was regular and synchronized bilaterally and ipsilaterally with the hypoglossal or cervical C1-C6 rootlets. Brainstem transection experiments demonstrated that for both the trigeminal and facial rootlets, the spontaneous rhythmic activity originates from the medulla, in a region consistent with the pre-Bötzinger complex and the rostral ventrolateral medulla. The pattern of the respiratory motor activity recorded from the trigeminal and facial rootlets was identical to the pattern recorded from the hypoglossal and cervical C1-C6 rootlets with rapidly peaking, slowly decrementing characteristics. The duration of the ascending part and the total duration of their active phases were similar. The onset of the active phases of the phrenic rootlets was delayed compared with that of the trigeminal, facial and hypoglossal rootlets. However, no difference in the onsets of the active phases of the cranial rootlets could be observed. Removal of the rostral pons suppressed the delay in onset of the active phases of the phrenic rootlets. Our findings show that: (i) rhythmic activities of the trigeminal and facial rootlets are preserved in absence of control by peripheral or high cerebral structures; (ii) the pattern and the location of the rhythmic generator for these activities are of the respiratory type; and (iii) the rostral pons is responsible for a delay in the onset of the active phases of the phrenic rootlets compared with that of the trigeminal, facial and hypoglossal rootlets.

K+ channels of expiratory neurones in the brain stem preparation of the newborn rat.

Single channel activity of expiratory neurones was studied in outside-out recordings. Expiratory neurones were identified in the ventrolateral region of the in vitro isolated brain stem-spinal cord preparation of newborn rats in cell-attached and whole-cell configurations by their pattern of firing related to phrenic motor output. Three potassium (K+) channels of 10, 30 and 70 pS exhibited steady-state activity during long voltage commands (up to 5 min) and could be found associated together in the same patches. The 30pS channel showed voltage dependency, being most active at small depolarizations. The 70 pS channel showed little activity with < 1% of openings per sample time and 1 mM tetraethylammonium (TEA) sensitivity. At similar concentrations, the discharge of the phrenic nerve was also altered, as shown by the increase of the respiratory frequency and a tonic discharge. The association of these K+ channel types on the same patches may be specific of respiratory neurones and could contribute to their bursting activity.

Reorganization of pontine rhythmogenic neuronal networks in Krox-20 knockout mice.

We have shown previously that the inactivation of the zinc finger gene Krox-20 affects hindbrain segmentation, resulting in the elimination of rhombomeres 3 and 5. We demonstrate here that Krox-20 homozygous mutant mice exhibit abnormally slow respiratory and jaw opening rhythms, indicating that a modification of hindbrain segmentation influences the function of neuronal networks after birth. Central neuronal networks that control respiratory frequency are made predominantly depressant by the elimination of a previously undescribed rhythm-promoting system. Recordings of rhythmic activity from the isolated hindbrain following progressive tissue transections indicate that the reorganization takes place in the caudal pontine reticular formation. The newborn (PO) Krox-20-/- mice, in which apneas are ten times longer than in wild-type animals, may be a valuable model for the study of life-threatening apneas during early infancy.

Substance P immunoreactivity of rat brain stem neurons in primary culture.

Substance P (SP)-ergic neurons from 16/17 day-embryonic rat brain stem in primary culture were identified by immunocytochemistry using biotinylated avidin and phosphatase alkaline methods with affinity-purified anti-SP antibodies. An average of 84% of neurons contained SP from day 9 to day 21 after plating. These in vitro data show that SP-containing neurons develop in our culture conditions. SP may act as a maturation factor as well as a neurotransmitter.