Cellular mechanisms for amyloid beta-protein activation of rat cholinergic basal forebrain neurons.

The deposition of amyloid beta-protein (Abeta) in the brain and the loss of cholinergic neurons in the basal forebrain are two pathological hallmarks of Alzheimer's disease (AD). Although the mechanism of Abeta neurotoxicity is unknown, these cholinergic neurons display a selective vulnerability when exposed to this peptide. In this study, application of Abeta(25-35) or Abeta(1-40) to acutely dissociated rat neurons from the basal forebrain nucleus diagonal band of Broca (DBB), caused a decrease in whole cell voltage-activated currents in a majority of cells. This reduction in whole cell currents occurs through a modulation of a suite of potassium conductances including calcium-activated potassium (I(C)), the delayed rectifier (I(K)), and transient outward potassium (I(A)) conductances, but not calcium or sodium currents. Under current-clamp conditions, Abeta evoked an increase in excitability and a loss of accommodation in cholinergic DBB neurons. Using single-cell RT-PCR technique, we determined that Abeta actions were specific to cholinergic, but not GABAergic DBB neurons. Abeta effects on whole cell currents were occluded in the presence of membrane-permeable protein tyrosine kinase inhibitors, genistein and tyrphostin B-44. Our data indicate that the Abeta actions on specific potassium conductances are modulated through a protein tyrosine kinase pathway and that these effects are selective to cholinergic but not GABAergic cells. These observations provide a cellular basis for the selectivity of Abeta neurotoxicity toward cholinergic basal forebrain neurons that are at the epicenter of AD pathology.

Increased neuromuscular activity reduces sprouting in partially denervated muscles.

The effects of increasing neural activity on sprouting remain unclear and controversial. In a rat model of partial denervation of skeletal muscles, we investigated the effect of neuromuscular activity on sprouting. Rat hindlimb muscles were partially denervated by avulsion of either L4 or L5 spinal root. Immediately after partial denervation, the rats were divided into three groups: (1) normal caged activity, (2) running exercise on wheels, 8 hr daily, and (3) functional electrical stimulation (FES) of sciatic nerves, 20 Hz for 8 hr daily. At 1 month, muscle unit (MU) enlargement was quantitated electrophysiologically and histochemically. MU twitch force was increased by four- to fivefold by partial denervation in extensively denervated tibialis anterior (TA) and medial gastrocnemius (MG) and by approximately twofold in moderately denervated plantaris (PL) and soleus (SOL). For the extensively denervated TA and MG muscles, MU enlargement, measured electrophysiologically, declined significantly after an average of 1757 +/- 310 m/d running exercise and daily FES for 1 month. The detrimental effects on MU enlargement were much less but significant in the moderately denervated PL and did not reach statistical significance in the moderately denervated SOL muscle. Histochemical evaluation of sprouting showed a reduction in the number of sprouts in the extensively denervated TA muscle, but not the moderately denervated PL and SOL muscles, by increased neuromuscular activity. Thus, increased neuromuscular activity is detrimental primarily in muscles that are extensively denervated, and the MUs are smaller than under conditions in which the muscles experience normal physiological levels of activation.

Zinc modulation of ionic currents in the horizontal limb of the diagonal band of Broca.

We examined modulation of ionic currents by Zn2+ in acutely dissociated neurons from the rat's horizontal limb of the diagonal band of Broca using the whole-cell patch-clamp technique. Application of 50 microM Zn2+ increased the peak amplitude of the transiently activated potassium current, I(A) (at + 30 mV), from 2.20+/-0.08 to 2.57+/-0.11 nA (n = 27). This response was reversible and could be repeated in 0 Ca2+/1 microM tetrodotoxin (n = 15). Zn2+ shifted the inactivation curve to the right, resulting in a shift in the half-inactivation voltage from 76.4+/-2.2 to -53.4+/-2.0 mV (n = 11), with no effect on the voltage dependence of activation gating (n = 15). There was no significant difference in the time to peak under control conditions (7.43+/-0.35 ms, n = 14) and in the presence of Zn2+ (8.20+/-0.57 ms, n = 14). Similarly, the time constant of decay of I(A) (tau(d)) at + 30 mV showed no difference (control: 38.68+/-3.68 ms, n = 15; Zn2+: 38.48+/-2.85 ms, n = 15). I(A) was blocked by 0.5-1 mM 4-aminopyridine. In contrast to its effects on I(A), Zn2+ reduced the amplitude of the delayed rectifier potassium current (I(K)). The reduction of outward K+ currents was reproducible when cells were perfused with 1 microM tetrodotoxin in a 0 Ca2+ external solution. The amplitude of the steady-state outward currents at +30 mV under these conditions was reduced from 6.40+/-0.23 (control) to 5.76+/-0.18 nA in the presence of Zn2+ (n = 16). The amplitudes of peak sodium currents (INa) were not significantly influenced (n = 10), whereas barium currents (I(Ba)) passing through calcium channels were potently modulated. Zn2+ reversibly reduced I(Ba) at -10 mV by approximately 85% from -2.06+/-0.14 nA under control conditions to -0.30+/-0.10 nA in the presence of Zn2+ (n = 14). Further analyses of Zn2+ effects on specific calcium channels reveals that it suppresses all types of high-voltage-activated Ca2+ currents. Under current-clamp conditions, application of Zn2+ resulted in an increase in excitability and loss of accommodation (n = 13), which appears to be mediated through its effects on Ca2+-dependent conductances.

Ionic mechanisms of action of neurotensin in acutely dissociated neurons from the diagonal band of Broca of the rat.

Whole cell recordings were performed on acutely dissociated neurons from the horizontal limb of the diagonal band of Broca (hDBB) from rats to elucidate the ionic mechanisms of action of neurotensin. Neurotensin caused a decrease in whole cell voltage-activated outward currents and failed to elicit a response when Ca2+ influx was blocked by changing the external solution to the one containing 0 mM Ca2+ and 50 microM Cd2+, suggesting the involvement of Ca2+-dependent conductances. Charybdotoxin, a specific blocker of voltage-sensitive calcium-activated K+ channels (IC), caused a decrease in outward currents comparable with that caused by blocking calcium influx and occluded the neurotensin-induced decrease in outward currents. Similarly, 50 microM tetraethylammonium ions also blocked the neurotensin response. Also neurotensin reduced whole cell barium currents (IBa) and calcium currents (ICa). Amiloride and omega-conotoxin GVIA, but not nimodipine, were able to eliminate the neurotensin-induced decrease in IBa. Thus T- and N- but not L-type calcium channels are subject to modulation by neurotensin, and this may account for its effects on IC. The predicted changes in action potential as a result of the blockade of currents through calcium channels culminating into changes in IC were confirmed in the bridge current-clamp recordings. Specifically, neurotensin application led to depolarization of the resting membrane potential, broadening of spike and a decrease in afterhyperpolarization and accommodation. These alterations in action potential characteristics that resulted in increased firing rate and excitability of the hDBB neurons also were produced by application of charybdotoxin. Neurotensin effects on these properties were occluded by 2 - [(1 - 7 - chloro - 4 - quinolinyl) - 5 - (2, 6 - di - methoxyphenyl) pyrazol-3-yl) carbonylamino] tricyclo (3.3.1.1.)decan-2-carboxylic acid, a nonpeptide high-affinity neurotensin receptor antagonist. Neurotensin blockade of IC, possibly through ICa, is a potential physiological mechanism whereby this peptide may evoke alterations in the cortical arousal, sleep-wake cycle, and theta rhythm.

Vasopressin receptor subtypes differentially modulate calcium-activated potassium currents in the horizontal limb of the diagonal band of Broca.

The actions of vasopressin on acutely dissociated neurons within the rat horizontal limb of the diagonal band of Broca were examined using the whole-cell patch-clamp technique. Vasopressin elicited two distinct responses in 45 of 62 neurons. In one group of cells, 300 nM vasopressin decreased voltage-activated outward currents (26/45 cells) whereas in a second group, vasopressin increased outward currents (19/45 cells). The vasopressin-mediated decrease in outward currents was blocked by 1 microM Manning compound, a V1 receptor antagonist, suggesting that this response was mediated via V1 receptors. In contrast, the vasopressin-induced increase in outward current was blocked by 1 microM d(CH2)5)1,D-Ile2,Ile4,Arg8,Ala9, a V2 receptor antagonist, indicating that V2 receptor activation underlies this second response. When cells were perfused with 0 Ca2+/50 microM Cd2+, application of vasopressin did not cause any change in voltage-activated outward currents, suggesting that vasopressin modulates a calcium-dependent conductance. In the presence of 25 nM charybdotoxin, an Ic channel antagonist, vasopressin application did not influence outward currents, indicating that vasopressin modulates Ic. Currents through voltage-gated calcium channels which are responsible for activation of Ic were unaffected by vasopressin, suggesting a direct effect of vasopressin on Ic channels. These observations indicate a differential modulation of Ic channels by vasopressin via V1 and V2 receptors in the horizontal limb of the diagonal band of Broca. Our data also demonstrate the ionic mechanisms whereby vasopressin may act at V1 for V2 receptors to influence the excitability of the horizontal limb of the diagonal band of Broca neurons.

GABAA receptor modulation by protein tyrosine kinase in the rat diagonal band of Broca.

The diagonal band of Broca (DBB) is involved in a wide array of physiological functions which are, in part, mediated by activation of GABAA receptors. DBB is enriched in GABA and protein tyrosine kinase (PTK) immunoreactivity. Whole-cell patch-clamp recording were performed from acutely dissociated DBB neurons to investigate the involvement of PTK in GABAA receptor function. The activation of GABAA receptor by the selective agonist, muscimol (5 microM) was dependent on the presence of intracellular ATP. Omission of ATP in the intracellular medium resulted in a fast decrement of the response whereas inclusion of sodium orthovanadate (100 microM), a non-specific phosphatase inhibitor, augmented the response and inhibited 'run down' of the response. Genistein (100 microM) and tyrphostin B-44 (-), specific inhibitors of PTK, attenuated the response to muscimol. The muscimol response was not affected by daidzein (100 microM); an inactive analogue of genistein) nor by tetraethylammonium bromide (1 mM). These observations suggest that phosphorylation is important for the activation and long term maintenance of GABAA receptor function. PTK phosphorylation, which has been previously identified as an important event in signal transduction, may modulate GABA mediated neurotransmission in the forebrain.

Phorbol ester-induced M-current suppression in bull-frog sympathetic ganglion cells: insensitivity to kinase inhibitors.

1. The effects of 1-oleoyl-2-acetyl-sn-glycerol (OAG), phorbol 12-myristate 13-acetate (PMA), 4-alpha-phorbol and muscarine on B-neurones from bull-frog sympathetic ganglion were studied by means of whole-cell patch-clamp recording. With the exception of 4-alpha-phorbol, all of these agonists reduced the steady-state outward current recorded at -30 mV as a result of suppression of a voltage-dependent, non-inactivating K(+)-current, the M-current, (IM). 2. Of the cells tested, 34% displayed bona fide responses to OAG (20 microM). The chance of recording a response was not decreased when the protein kinase inhibitor, 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7; 50 or 75 microM) was included simultaneously in the extracellular solution and in the pipette solution. 3. The presence of 50 microM H-7 on both sides of the membrane or 500 nM staurosporine in the pipette solution did not prevent responses to brief (1-2 min) or prolonged (> 20 min) applications of PMA. 4. Brief (1-2 min) extracellular application of H-7 (300 microM) suppressed IM by about 29%. 5. The most likely explanation of these data is that PMA and OAG modulate IM via a mechanism that is independent of protein kinase C (PKC). The availability of such a mechanism poses new questions as to the mechanism of muscarine-induced IM suppression.

Changes in potassium channel activity following axotomy of B-cells in bullfrog sympathetic ganglion.

1. Whole-cell and microelectrode voltage-clamp techniques were used to investigate the changes in ionic currents and action potential shape that follow axotomy of bullfrog paravertebral sympathetic ganglion B-cells. 2. Axotomy increased M-conductance (gM; muscarine-sensitive, voltage- and time-dependent K+ conductance) by 35% at -30 mV and slowed its deactivation kinetics. 3. The delayed rectifier K+ current (IK; at +50 mV) was reduced in axotomized neurones to 61% of control without any change in activation or deactivation kinetics. Steady-state intracellular Ca2+ levels and leak conductance were unchanged. 4. The fast, voltage-sensitive, Ca(2+)-activated K+ current (IC), evoked from -40 mV, was decreased to about 71% of control (at +30 mV) in axotomized neurones, whereas that evoked from -80 mV was largely unaffected. IC kinetics were also similar in control and axotomized neurones. This suggests that IC channels are not changed after axotomy. 5. In axotomized neurones, commands to +10 from -40 mV had to be extended by 16 ms to evoke voltage-insensitive Ca(2+)-dependent K+ current (IAHP) responses that were similar in magnitude to those observed in control cells. 6. The previously documented, axotomy-induced decrease in Ca2+ current (ICa) due to increased resting inactivation can account for the reduction in IC and IAHP and for the change in the shape of the action potential.

Changes in sodium and calcium channel activity following axotomy of B-cells in bullfrog sympathetic ganglion.

1. Currents mediated by Ca2+ channels using Ba2+ as a charge carrier (IBa), Na+ currents (INa) and voltage- and Ca(2+)-dependent K+ currents (IC) were recorded from bullfrog paravertebral sympathetic ganglion B-cells using whole-cell patch-clamp recording techniques. Currents recorded from control cells were compared with those from axotomized cells 13-15 days after transection of the postganglionic nerve. 2. Axotomy reduced peak IBa at -10 mV (holding potential = -80 mV) from 3.3 +/- 0.3 nA (n = 42) to 1.7 +/- 0.1 nA (n = 39, P < 0.001). Tail IBa at -40 mV following a step to +70 mV from a holding potential of -80 mV was also reduced in axotomized neurones (9.7 +/- 0.6 nA for forty-two control neurones and 5.2 +/- 0.3 nA for thirty-nine axotomized neurones; P < 0.001). Minimal changes were observed in the kinetics of activation and deactivation. 3. Pharmacological experiments using 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2- trifluoromethylphenyl)-pyridine-5-carboxylic acid methyl ester (BayK 8644), nifedipine and omega-conotoxin showed that axotomy predominantly affected the N-type Ca2+ channels which carry the majority of ICa in these neurones. L-type Ca2+ current was little affected and T-type Ca2+ currents were not observed in control or axotomized cells. 4. Development of inactivation of 0 mV and recovery from inactivation of IBa at -80 mV exhibited three distinct components in both control and axotomized neurones: 'fast', 'intermediate' and 'slow'. The relative proportions of both the 'fast' and 'intermediate' components of inactivation at 0 mV were almost doubled after axotomy (fast component was 15% in control and 29% in axotomized neurones; intermediate component was 17% in control and 26% in axotomized neurones). 'Fast' and 'intermediate' inactivation tended to develop more rapidly and recover more slowly after axotomy. The rate of onset of 'slow' inactivation was unaffected by axotomy but the steady-state level at -40 mV was increased. Most of the change in IBa properties may be secondary to enhanced inactivation associated with axotomy. 5. Axotomy reduced IC (measured at the end of a 3 ms step from -40 to +20 mV) from 34.5 +/- 4.9 (n = 26) to 19.2 +/- 1.5 nA (n = 49, P < 0.005). This reduction may be secondary to the reduction in calcium channels available for activation from -40 mV following axotomy. 6. The TTX-sensitive and TTX-insensitive components of peak Na+ conductance (GNa) were both increased after axotomy. Total GNa was increased from 184.9 +/- 8.4 to 315.2 +/- 16.4 nS (n = 37 for both P < 0.001). Most of the kinetic and steady-state properties of INa were unchanged after axotomy.(ABSTRACT TRUNCATED AT 400 WORDS)

Slow frequency-dependence of action potential afterhyperpolarization in bullfrog sympathetic ganglion neurones.

The after hyperpolarizatin (AHP) which follows the action potential (AP) in bullfrog sympathetic ganglion B-cells involves activation of Ca(2+)-sensitive K+ conductances following Ca2+ influx via Ca2+ channels. The duration of AHPs evoked at 2-s stimulus intervals were 70.05 +/- 3.76% of those evoked at 90-s stimulus intervals (n = 35). Since there was no consistent effect of ryanodine (5 microM), ruthenium red, (300 microM) or dantrolene Na (35 microM) on this frequency dependence, it is unlikely to result from release of Ca2+ from intracellular stores. Ca2+ currents (ICa), studied by means of the whole-cell patch-clamp technique, exhibited a slow frequency dependence as a result of a slow inactivation process which was independent of Ca(2+)-induced ICa inactivation and ICa run-down. There was excellent correlation (r = 0.964) between the estimated changes in Ca2+ influx and the expected activation of the Ca(2+)-sensitive K+ current, IAHP. This result is consistent with the hypothesis that the frequency dependence of the AHP is a consequence of the slow inactivation of ICa.