SUMMARY1. Developmental changes in acetylcholine (ACh) receptor channel function on aneural cultures of embryonic myotomal muscle cells were examined using the patch-clamp technique.2. At all stages of differentiation two different unitary-event amplitudes were observed, corresponding to high-y (single-channel conductance) (64 pS) and low-y (46 pS) channel types. No change in conductance occurred for either channel type during the 6-day in vitro period examined.3. At resting membrane potential (-85 mV) the low-y channel exhibited a mean open time of approximately 2 ms which, on the average, was 2-3-fold longer than that measured for the high-y channel. Neither the estimated mean channel open time nor the voltage dependence of the open state measured for either channel type changed during development.4. In recordings with low ACh concentration (041-0-25 ,SM) both high-y and low-y channel types exhibited non-stationary opening probabilities over the recording period. Usually the opening rate of both channel types decreased with time following seal formation, however, the 'drop-out' rate was faster for the low-y channel.5. A developmental increase in the proportion of high-y events occurred between day 1 (16 %) and day 5 (56 %) in culture, paralleling the time-dependent changes in the channel kinetics based on ACh-activated membrane noise.6. We conclude that the development of non-junctional muscle membrane is associated with increased expression of high-y channels and that this process is primarily responsible for the previously reported developmental alterations in macroscopic ACh receptor channel currents.

SUMMARY1. Patch-clamp recordings of current through acetylcholine-activated channels were made from non-junctional membrane of innervated myotomal muscle from Xenopus laevis.2. Two classes ofacetylcholine (ACh) receptor channels were identified on the basis of current amplitudes. Both amplitude classes exhibited current-voltage relations which deviated from linearity as the extrapolated reversal potential was approached (-5 to -12 mV). Over the range of greatest linearity the conductances of the two classes were 64 and 44 pS. Both event classes were blocked by ac-bungarotoxin. 5. In the absence ofexogenous ACh, channels were observed in an occasional patch which showed a conductance and extrapolated reversal potential similar to AChactivated channels. In such patches the event frequency could occasionally be altered by adjusting the negative pressure applied to the patch.6. The two main conductance classes of ACh activated channels were observed to coexist in most patches. However, the most frequent event observed in non-junctional membrane of innervated muscle corresponded to the high y class. In this respect, the non-junctional ACh receptors bore a greater similarity to junctional ACh receptors than to non-junctional receptors reported for denervated muscle.

Dementia is a major cause of morbidity in the western society. Pharmacological therapies to delay the progression of cognitive impairments are modestly successful. Consequently, new therapies are urgently required to improve cognitive deficits associated with dementia. We evaluated the effects of physical and cognitive activity on learning and memory in a rat model of vascular dementia (VasD). Male Sprague-Dawley rats (6 months old) were exposed to either regular chow or a diet rich in saturated fats and sucrose and chronic bilateral common carotid artery occlusion or sham surgery. First, this model of VasD was validated using a 2  2 experimental design (surgery  diet) and standard cognitive outcomes. Next, using identical surgical procedures, we exposed animals to a paradigm of cognitive rehabilitation or a sedentary condition. At 16 weeks post surgery, VasD animals demonstrated significant learning and memory deficits in the Morris water maze, independent of diet. Rehabilitation significantly attenuated these cognitive deficits at this time point as well as at 24 weeks. Further, rehabilitation normalized hippocampal CA1 soma size (area and volume) to that of control animals, independent of cell number. Importantly, these findings demonstrate beneficial neuroplasticity in early middle-aged rats that promoted cognitive recovery, an area rarely explored in preclinical studies.

Embryonic Xenopus muscle cells grown in culture develop discrete patches of high acetylcholine receptor (AChR) density. By following identified muscle cells after staining with fluorescent alpha-bungarotoxin, we have found that many of these AChR patches survive in a fixed position for several days. For AChR patches on the lower surface of the cell (the surface apposed to the culture dish), more than 60% of those which were followed beginning on day 2 survived for a further 4 days. The survival rate was greater when patches were followed from day 3 or later and was almost as high for AChR patches on the upper surface. New AChR patches also formed on all of the muscle cells. When muscle cells were cultured together with spinal cord cells, nerve-muscle contacts developed with a characteristic localization of AChRs along the path of contact. AChR patches did not form elsewhere on these contacted cells. Nerve-contacted muscle cells examined 2 to 3 days after adding spinal cord cells to established (2- to 5-day-old) muscle cultures also exhibited a marked reduction of AChR patches away from the site of contact. This reduction was not due to the nerve having contacted pre-existing AChR patches. Rather, the findings indicate that contact by an appropriate nerve inhibits the formation of AChR patches elsewhere on the contracted muscle cells and reduces the survival of pre-existing AChR patches. Nerve contact also inhibited the formation of cholinesterase (ChE) patches remote from the site of contact and appeared to cause some reduction in the survival of pre-existing ChE patches. Spontaneous twitching was not observed in these experiments, thereby indicating that the remote effects of nerve contact were not mediated by muscle action potentials or contraction. Such remote influences of the nerve may play a role in determining the pattern of innervation on individual muscle cells.

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Recent experiments have indicated that neural agrin is deposited at newly forming nerve-muscle synapses and has a primary synaptogenic role there. As a step toward assessing how the spatial arrangement of new synaptic sites is regulated, we compared the pattern of agrin deposition by Xenopus neurites on culture substrate and on muscle cells. The neurons were grown on a substrate that bound their externalized agrin so tightly that it remained bound even when the neurites retracted spontaneously or were eliminated experimentally. By contrast, the neural cell adhesion molecule, NCAM, was not left behind on the substrate when the neurites were eliminated. Agrin, visualized by immunofluorescent staining, was deposited on the culture substrate in a continuous fashion along virtually the entire neuritic arbor of many spinal cord (SC) neurites. The pattern of agrin deposition by the same neurites changed from continuous to discontinuous when the neurites contacted muscle cells, and it became continuous again when the neurites returned to the culture substrate. The sites of agrin deposition on muscle cells were also sites of accumulation of ACh receptors (AChRs). Dorsal root ganglion (DRG) neurons and some SC neurons did not deposit agrin along their neuritic outgrowth, either on the culture substrate or on the muscle cells, and did not induce AChR accumulation at sites of contact with muscle cells. Besides adding to the evidence in support of agrin's synaptogenic role, the findings indicate that muscle cells significantly influence how neural agrin and synaptic sites become distributed along paths of neurite-muscle contact.

Myotomal muscle cells, derived from Xenopus embryos and grown in culture without nerve, develop discrete sites of cholinesterase (ChE) activity on their surface. The spatial relationship of these ChE patches to surface patches of acetylcholine receptors (AChRs) has been examined in the present study by a combination of ChE histochemistry and fluorescent staining of the receptors. ChE patches and AChR patches developed as early as the 1st day in culture and exhibited a high incidence of spatial overlap. The frequency of overlap varied with the age of the culture and ranged from 50 to 98% for patches on the lower cell surface (facing the floor of the culture dish) and from 28 to 79% for patches on the upper cell surface. The high incidence of overlap cannot be explained on the basis of a random distribution of patches since both types of patch occupied less than 3% of the cell surface. ChE and AChR patches also developed when cultures were grown in a serum-free medium as well as when cultures were prepared from young embryos in which muscle innervation had not yet begun. At some patches, the surface membrane was invaginated and at these invaginations, there was also a high incidence of overlap between the ChE and AChR stains.It is concluded that the mechanisms involved in the localization of AChRs and ChE on the surface of Xenopus myotomal muscle cells tend to be closely linked and operate even in the absence of innervation, previous contact by nerve, or electrical and contractile activity. Considered together with previous ultrastructural observations, the present findings suggest that these cells develop elaborate postsynaptic-like specializations even in the absence of neural factors.

Background and Purpose-Recent studies show that prolonged (eg, 24-hour) postischemic hypothermia confers lasting histological and behavioral protection against severe global cerebral ischemia. However, functional abnormalities may be compensated for by undamaged brain regions and thus not detected by behavioral tests. To determine whether hypothermia preserves CA1 functional integrity, we measured synaptic and membrane properties of CA1 neurons in ischemic gerbils treated with postischemic hypothermia. Methods-Gerbils were subjected to 5 minutes of forebrain ischemia and were either left untreated or exposed to 2 days of hypothermia (32°C for 24 hours and then 34°C for 24 hours). Sham animals were operated on but not made ischemic, then either allowed to recover at room temperature or subjected to hypothermia for 2 days. Approximately 5 weeks after ischemia or sham surgery, patch-clamp recordings were obtained from the CA1 region of hippocampal slices. Results-There was approximately 95% CA1 cell loss in untreated ischemic animals, whereas ischemic gerbils treated with hypothermia had cell counts similar to sham animals. Resting membrane potential, action potential amplitude and duration, input resistance, and synaptic currents evoked by Schaffer collateral stimulation were similar between pyramidal cells obtained from ischemic gerbils treated with hypothermia and sham-operated animals (PϾ0.05). Conclusions-These data demonstrate that postischemic hypothermia preserves the measured electrophysiological properties of CA1 neurons in the absence of any apparent functional abnormalities. This study provides further support for the use of hypothermia as a treatment for cerebral ischemia.