David A. Prince

Mechanisms of action of acetylcholine in the guinea-pig cerebral cortex in vitro.

The mechanisms of action of acetylcholine (ACh) in the guinea‐pig neocortex were investigated using intracellular recordings from layer V pyramidal cells of the anterior cingulate cortical slice. At resting membrane potential (Vm = ‐80 to ‐70 mV), ACh application resulted in a barrage of excitatory and inhibitory post‐synaptic potentials (p.s.p.s) associated with a decrease in apparent input resistance (Ri). ACh, applied to pyramidal neurones depolarized to just below firing threshold (Vm = ‐65 to ‐55 mV), produced a short‐latency hyperpolarization concomitant with p.s.p.s and a decrease in Ri, followed by a long‐lasting (10 to greater than 60 s) depolarization and action potential generation. Both of these responses were also found in presumed pyramidal neurones of other cortical regions (sensorimotor and visual) and were blocked by muscarinic, but not nicotinic, antagonists. The ACh‐induced hyperpolarization possessed an average reversal potential of ‐75.8 mV, similar to that for the hyperpolarizing response to gamma‐aminobutyric acid (GABA; ‐72.4 mV) and for the i.p.s.p. generated by orthodromic stimulation (‐69.6 mV). This cholinergic inhibitory response could be elicited by ACh applications at significantly greater distance from the cell than the slow depolarizing response. Blockade of GABAergic synaptic transmission with solution containing Mn2+ and low Ca2+, or by local application of tetrodotoxin (TTX), bicuculline or picrotoxin, abolished the ACh‐induced inhibitory response but not the slow excitatory response. In TTX (or Mn2+, low Ca2+) the slow excitatory response possessed a minimum onset latency of 250 ms and was associated with a voltage‐dependent increase in Ri. Application of ACh caused short‐latency excitation associated with a decrease in Ri in eight neurones. The time course of this excitation was similar to that of the inhibition seen in pyramidal neurones. Seven of these neurones had action potentials with unusually brief durations, indicating that they were probably non‐pyramidal cells. ACh blocked the slow after‐hyperpolarization (a.h.p.) following a train of action potentials, occasionally reduced orthodromically evoked p.s.p.s, and had no effect on the width or maximum rate of rise or fall of the action potential. It is concluded that cholinergic inhibition of pyramidal neurones is mediated through a rapid muscarinic excitation of non‐pyramidal cells, resulting in the release of GABA. In pyramidal cells ACh causes a relatively slow blockade of both a voltage‐dependent hyperpolarizing conductance (M‐current) which is most active at depolarized membrane potentials, and the Ca2+‐activated K+ conductance underlying the a.h.p.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel T-type current underlies prolonged Ca(2+)-dependent burst firing in GABAergic neurons of rat thalamic reticular nucleus

The inhibitory GABAergic projection of thalamic nucleus reticularis (nRt) neurons onto thalamocortical relay cells (TCs) is important in generating the normal thalamocortical rhythmicity of slow wave sleep, and may be a key element in the production of abnormal rhythms associated with absence epilepsy. Both TCs and nRt cells can generate prominent Ca(2+)-dependent low-threshold spikes, which evoke bursts of Na(+)-dependent fast spikes, and are influential in rhythm generation. Substantial differences in the pattern of burst firing in TCs versus nRt neurons led us to hypothesize that there are distinct forms of transient Ca2+ current (I(T)) underlying burst discharges in these two cell types. Using whole-cell voltage-clamp recordings, we analyzed I(T) in acutely isolated TCs and nRt neurons and found three key differences in biophysical properties. (1) The transient Ca2+ current in nRt neurons inactivated much more slowly than I(T) in TCs. This slow current is thus termed I(Ts). (2) The rate of inactivation for I(Ts) was nearly voltage independent. (3) Whole-cell I(Ts) amplitude was increased when Ba2+ was substituted for Ca2+ as the charge carrier. In addition, activation kinetics were slower for I(Ts) and the activation range was depolarized compared to that for I(T). Other properties of I(Ts) and I(T) were similar, including steady-state inactivation and sensitivities to blockade by divalent cations, amiloride, and antiepileptic drugs. Our findings demonstrate that subtypes of transient Ca2+ current are present in two different classes of thalamic neurons. The properties of I(Ts) lead to generation of long-duration calcium-dependent spike bursts in nRt cells. The resultant prolonged periods of GABA release onto TCs would play a critical role in maintaining rhythmicity by inducing TC hyperpolarization and promoting generation of low-threshold calcium spikes within relay nuclei.

Calcium currents in rat thalamocortical relay neurones: kinetic properties of the transient, low-threshold current.

1. Calcium currents were recorded with whole‐cell voltage‐clamp procedures in relay neurones of the rat thalamus which had been acutely isolated by an enzymatic dissociation procedure. 2. Low‐threshold and high‐threshold Ca2+ currents were elicited by depolarizing voltage steps from holding potentials more negative than ‐60 mV. A transient current, analogous to the T‐current in sensory neurones, was activated at low threshold near ‐65 mV and was completely inactivating at command steps up to ‐35 mV. Voltage steps to more depolarized levels activated a high‐threshold current that inactivated slowly and incompletely during a 200 ms step depolarization. 3. The high‐threshold current contained both non‐inactivating and slowly inactivating components which were insensitive and sensitive to holding potential, respectively. 4. A ‘T‐type’ current was prominent in relay neurones, in both absolute terms (350 pA peak current average) and in relation to high‐threshold currents. The average ratio of maximum transient to maximum sustained current was greater than 2. 5. T‐current could be modelled in a manner analogous to that employed for the fast Na+ current underlying action potential generation, using the m3h format. The rate of activation of T‐current was voltage dependent, with a time constant (tau m) varying between 8 and 2 ms at command potentials of ‐60 to ‐10 mV at 23 degrees C. The rate of inactivation was also voltage dependent, and the time constant tau h varied between 50 and 20 ms over the same voltage range. With command potentials more positive than ‐35 mV, the inactivation of Ca2+ current could no longer be fitted by a single exponential. 6. Steady‐state inactivation of T‐current could be well fitted by a Boltzman equation with slope factor of 6.3 and half‐inactivated voltage of ‐83.5 mV. 7. Recovery from inactivation of T‐current was not exponential. The major component of recovery (70‐80% of total) was not very voltage sensitive at potentials more negative than ‐90 mV, with tau r of 251 ms at ‐92 mV and 23 degrees C, compared to 225 ms at ‐112 mV. A smaller, voltage‐sensitive component accounted for the remainder of recovery. 8. All kinetic properties, including rates of activation, inactivation, and recovery from inactivation, as well as the amplitude of T‐current, were temperature sensitive with Q10 (temperature coefficient) values of greater than 2.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Frequency‐dependent depression of inhibition in guinea‐pig neocortex in vitro by GABAB receptor feed‐back on GABA release.

1. The mechanisms involved in the lability of inhibition at higher frequencies of stimulation were investigated in the guinea‐pig in vitro neocortical slice preparation by intracellular recording techniques. We attempted to test the possibility of a feedback depression of GABA on subsequent release. 2. At resting membrane potential (Em, ‐75.8 +/‐ 5.2 mV) stimulation of either the pial surface or subcortical white matter evoked a sequence of depolarizing and hyperpolarizing synaptic components in most neurones. An early hyperpolarizing component (IPSPA) was usually only obvious as a pronounced termination of the EPSP, followed by a later hyperpolarizing event (IPSPB). Current‐voltage relationships revealed two different conductances of about 200 and 20 nS and reversal potentials of ‐73.0 +/‐ 4.4 and ‐88.6 +/‐ 6.1 mV for the early and late component, respectively. 3. The conductances of IPSPA and IPSPB were fairly stable at a stimulus frequency of 0.1 Hz. At frequencies between 0.5 and 2 Hz both IPSPs were attenuated with the second stimulus and after about five stimuli a steady state was reached. Concomitantly IPSPs were shortened. The average decrease in synaptic conductance between 0.1 and 1 Hz was 80% for the IPSPA and 60% for the IPSPB. At these frequencies the reversal potentials decreased by 5 and 2 mV, respectively; Em and input resistance (Rin) were not consistently affected. 4. The amplitudes of field potentials, action potentials and EPSPs of pyramidal cells were attenuated less than 10% at stimulus frequencies up to 1 Hz, suggesting that alterations in local circuits between the stimulation site and excitatory input onto inhibitory interneurones may play only a minor role in the frequency‐dependent decay of IPSPs. 5. Localized application of GABA produced multiphasic responses. With low concentrations and application near the soma an early hyperpolarization prevailed followed by a depolarizing late component. Brief application of GABA at low frequencies induced constant responses; at higher frequencies, the responses sometimes declined. The current‐voltage relationships of the two GABA responses were similar to each other and to the early IPSP. An apparently fivefold higher conductance was estimated at lower Ems, suggesting that the GABA response had a voltage sensitivity. The slope conductance of IPSPs was decreased by up to 50% for tens of seconds after postsynaptically detectable effects of GABA had dissipated. 6. Application of the GABA uptake inhibitor nipecotic acid (50‐500 microM) reduced the conductance of both components of orthodromically evoked inhibition and shortened the IPSP at low frequencies, but had no additional effects at higher stimulation rates.(ABSTRACT TRUNCATED AT 400 WORDS)

Actions of acetylcholine in the guinea-pig and cat medial and lateral geniculate nuclei, in vitro.

1. The mechanisms of action of acetylcholine (ACh) in the medial (m.g.n.) and dorsal lateral geniculate (l.g.n.d.) nuclei were investigated using intracellular recordings techniques in guinea‐pig and cat in vitro thalamic slices. 2. Application of ACh to neurones in guinea‐pig geniculate nuclei resulted in a hyperpolarization in all neurones followed by a slow depolarization in 52% of l.g.n.d. and 46% of m.g.n. neurones. Neither the hyperpolarization nor the slow depolarization were eliminated by blockade of synaptic transmission and both were activated by acetyl‐beta‐methylcholine and DL‐muscarine and blocked by scopolamine, indicating that these responses are mediated by direct activation of muscarinic receptors on the cells studied. 3. The ACh‐induced hyperpolarization was associated with an increase in apparent input conductance (Gi) of 4‐13 nS. The reversal potential of the ACh‐induced hyperpolarization varied in a Nernstian manner with changes in extracellular [K+] and was greatly reduced by bath application of the K+ antagonist Ba2+ or intracellular injection of Cs+. These findings show that the muscarinic hyperpolarization is mediated by an increase in K+ conductance. 4. The ACh‐induced slow depolarization was associated with a decrease in Gi of 2‐15 nS, had an extrapolated reversal potential near EK, and was sensitive to [K+]o, indicating that this response is due to a decrease in K+ conductance. 5. In contrast to effects on guinea‐pig geniculate neurones, applications of ACh to cat l.g.n.d. and m.g.n. cells resulted in a rapid depolarization in nearly all cells, followed in some neurones by a hyperpolarization and/or a slow depolarization. The rapid excitatory response was associated with an increase in membrane conductance, had an estimated reversal potential of ‐49 to ‐4 mV and may be mediated by nicotinic receptors. The hyperpolarization and slow depolarization were similar to those of the guinea‐pig in that they were associated with an increase and decrease, respectively, of Gi, and were mediated by muscarinic receptors. 6. The muscarinic hyperpolarization interacted with the intrinsic properties of the thalamic neurones to inhibit single‐spike activity while promoting the occurrence of burst discharges. The muscarinic slow depolarization had the opposite effect; it brought the membrane potential into the range where burst firing was blocked and single‐spike firing predominated. Depending upon the membrane potential, the rapid excitatory response of cat geniculate neurones could activate either a burst or a train of action potentials.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholinergic excitation of mammalian hippocampal pyramidal cells.

Responses of CA1 pyramidal neurons to ACh were recorded with intracellular microelectrodes utilizing the in vitro guinea pig hippocampal slice preparation. ACh was delivered by drop or iontophoretic application to stratum oriens or stratum radiatum. Threshold dose for drop application was 1 mM. An initial hyperpolarization of 3.1 +/- 1.8 (S.D.) mV associated with a decrease in membrane input resistance (RN) of 21 +/- 9% (S.D.) occurred in about half the cells. This result is consistent with a presynaptic action of ACh mediated through excitation of inhibitory interneurons. This interpretation was supported by recordings of cholinergic excitatory responses from presumed interneurons, and repetitive spontaneous IPSPs from pyramidal neurons during the hyperpolarization. ACh evoked a slow depolarization (14.3 +/- 10.8 (S.D.) mV) accompanied by a peak increase in apparent input resistance (Ra) of about 60% in the majority of cells. Large increases in spike frequency were associated with these events but action potential shape was unchanged. Plots of Ra versus membrane potential following ACh application revealed that Ra increases were proportionately higher at depolarized membrane potential levels (less than or equal to -70 mV) in some neurons. In these cells Ra was increased significantly at -60 mV (28%), but only 6% at -75 mV. These results are consistent with the conclusion that ACh reduces a voltage-dependent gK, distinct from delayed rectification. ACh also induced a non-voltage-dependent increase in Ra in some cells. ACh-evoked changes in Ra were long-lasting and gave rise to alterations in firing mode, with development of burst generation. ACh also transiently blocked after hyperpolarizations which followed spike trains in pyramidal neurons and presumed interneurons, an action which may be related to effects on a Ca2+-activated gK.

Post-natal development of electrophysiological properties of rat cerebral cortical pyramidal neurones.

1. The post‐natal development of the electrophysiological properties of cortical layer V pyramidal neurons was investigated with intracellular recordings from rat sensorimotor cortical slices, in vitro. 2. At all ages post‐natally (post‐natal day 1 to day 36; P1‐P36) neurons were capable of generating a train of Na+‐dependent action potentials in response to intracellular injection of sufficient depolarizing current. During the second and third week post‐natally, these action potentials changed substantially, becoming faster in both their rising and falling phases, shorter in duration, and larger in amplitude. 3. Both mature (greater than P21) and immature (P2‐P4) cortical neurones could generate Ca2+‐dependent action potentials only if a substantial portion of K+ conductances were blocked. The maximum rate of rise of Ca2+ spikes also increased with age. 4. The apparent input resistance, specific membrane resistance, and membrane time constant all decreased with age from P1 to P30. Immature neurones had I‐V relationships that were substantially more linear than those of adult cells, although rectification was often present in both the hyperpolarizing and depolarizing range. Inward rectification in the depolarizing range was Na+ dependent and was substantially larger in mature versus immature neurones. 5. Single, or trains of, action potentials in immature neurones were followed by short duration (10‐50 ms) and long duration (1‐5 s) after‐hyperpolarizations (a.h.p.s) respectively. The duration of the latter appeared to decrease with age. The presence of large a.h.p.s indicates that Ca2+ entry occurs during the action potential of immature, as well as mature, neurones. 6. Responses to intracellular injection of depolarizing current pulses indicated that immature neurones have frequency versus injected current (f‐I) relationships which are in general less steep than those for adult neurones and more limited in terms of the range of firing frequencies. 7. Our results are consistent with the hypothesis that there is a considerable increase in the density of voltage‐dependent ionic channels underlying the electro‐responsiveness of cortical pyramidal neurones during post‐natal development.

Extracellular potassium activity during epileptogenesis

Abstract Direct measurements of extracellular potassium concentration ([K + ] 0 ) changes in penicillin epileptogenic foci of cat cortex were made using potassium-sensitive microelectrodes. Interictal EEG events were accompanied by increases in [K + ] 0 lasting several seconds. The amplitudes and rise rates of these increases varied with cortical depth, distance from the center of the focus, and the [K + ] 0 at which they occurred. During ictal events, [K + ] 0 consistently reached 9–10 m m . The patterns of ictal [K + ] 0 changes also showed variations with depth in the cortex and distance from the focus. There was an upper limit for [K + ] 0 during both interictal and ictal epileptiform activity at about 10 m m . The role of these [K + ] 0 changes in modulating neuronal excitability in the epileptogenic focus is discussed. The [K + ] 0 level did not appear to be a critical factor in initiation or termination of ictal episodes.

Long-lasting self-inhibition of neocortical interneurons mediated by endocannabinoids

Neocortical GABA-containing interneurons form complex functional networks responsible for feedforward and feedback inhibition and for the generation of cortical oscillations associated with several behavioural functions. We previously reported that fast-spiking (FS), but not low-threshold-spiking (LTS), neocortical interneurons from rats generate a fast and precise self-inhibition mediated by inhibitory autaptic transmission. Here we show that LTS cells possess a different form of self-inhibition. LTS, but not FS, interneurons undergo a prominent hyperpolarization mediated by an increased K+-channel conductance. This self-induced inhibition lasts for many minutes, is dependent on an increase in intracellular [Ca2+] and is blocked by the cannabinoid receptor antagonist AM251, indicating that it is mediated by the autocrine release of endogenous cannabinoids. Endocannabinoid-mediated slow self-inhibition represents a powerful and long-lasting mechanism that alters the intrinsic excitability of LTS neurons, which selectively target the major site of excitatory connections onto pyramidal neurons; that is, their dendrites. Thus, modulation of LTS networks after their sustained firing will lead to long-lasting changes of glutamate-mediated synaptic strength in pyramidal neurons, with consequences during normal and pathophysiological cortical network activities.

Ionic mechanisms of cholinergic excitation in mammalian hippocampal pyramidal cells

Intracellular recordings from CA1 hippocampal pyramidal neurons were obtained using the in vitro hippocampal slice preparation. Responses to ACh were monitored in the presence of blockers of voltage-dependent conductances including Mn2+, TTX and Ba2+. When Mn2+ was used to block voltage-dependent Ca conductance and possible indirect presynaptic cholinergic actions, ACh still induced a significant voltage-sensitive increase in apparent input resistance (Ra) (29%), but only an insignificant depolarization of membrane potential (Vm). When both voltage-dependent Ca and Na conductances were blocked by application of Mn2+ and TTX, respectively, ACh produced voltage-dependent increases in Ra (31%) without significant depolarization. In solutions containing TTX alone, ACh produced voltage-sensitive increases in Ra (32%) as well as a significant depolarization (6.2 +/- 3.1 mV (S.D.)). ACh transiently blocked the conductance increase which followed presumed Ca spikes, suggesting an action on the Ca-activated K-dependent conductance. The effects of Ba2+ application (100-200 microM) on Ra mimicked those of ACh. When ACh was applied to neurons in the presence of Ba2+, Ra remained unchanged, although Vm depolarization of 5-15 mV was still seen. The data indicate that ACh decreases both a voltage-dependent K conductance (distinct from that of the delayed rectifier) and a Ca-activated K conductance. Muscarinic cholinergic depolarization occurs as a result of blockade of K conductance, and is mediated by voltage-dependent Ca and Na conductances, and perhaps by presynaptic actions.