Individual CA1 Pyramidal Neurons Research Articles

Effects of direct current stimulation on synaptic plasticity in a single neuron

BackgroundTranscranial direct current stimulation (DCS) has lasting effects that may be explained by a boost in synaptic long-term potentiation (LTP). We hypothesized that this boost is the result of a modulation of somatic spiking in the postsynaptic neuron, as opposed to indirect network effects. To test this directly we record somatic spiking in a postsynaptic neuron during LTP induction with concurrent DCS. MethodsWe performed rodent in-vitro patch-clamp recordings at the soma of individual CA1 pyramidal neurons. LTP was induced with theta-burst stimulation (TBS) applied concurrently with DCS. To test the causal role of somatic polarization, we manipulated polarization via current injections. We also used a computational multi-compartment neuron model that captures the effect of electric fields on membrane polarization and activity-dependent synaptic plasticity. ResultsTBS-induced LTP was enhanced when paired with anodal DCS as well as depolarizing current injections. In both cases, somatic spiking during the TBS was increased, suggesting that evoked somatic activity is the primary factor affecting LTP modulation. However, the boost of LTP with DCS was less than expected given the increase in spiking activity alone. In some cells, we also observed DCS-induced spiking, suggesting DCS also modulates LTP via induced network activity. The computational model reproduces these results and suggests that they are driven by both direct changes in postsynaptic spiking and indirect changes due to network activity. ConclusionDCS enhances synaptic plasticity by increasing postsynaptic somatic spiking, but we also find that an increase in network activity may boost but also limit this enhancement.

Switching between persistent firing and depolarization block in individual rat CA1 pyramidal neurons.

The hippocampal formation plays a role in mnemonic tasks and epileptic discharges in vivo. In vitro, these functions and malfunctions may relate to persistent firing (PF) and depolarization block (DB), respectively. Pyramidal neurons of the CA1 field have previously been reported to engage in either PF or DB during cholinergic stimulation. However, it is unknown whether these cells constitute disparate populations of neurons. Furthermore, it is unclear which cell-specific peculiarities may mediate their diverse response properties. However, it has not been shown whether individual CA1 pyramidal neurons can switch between PF and DB states. Here, we used whole cell patch clamp in the current clamp mode on in vitro CA1 pyramidal neurons from acutely sliced rat tissue to test various intrinsic properties which may provoke individual cells to switch between PF and DB. We found that individual cells could switch from PF to DB, in a cholinergic agonist concentration dependent manner and depending on the parameters of stimulation. We also demonstrate involvement of TRPC and potassium channels in this switching. Finally, we report that the probability for DB was more pronounced in the proximal than in the distal half of CA1. These findings offer a potential mechanism for the stronger spatial modulation in proximal, compared to distal CA1, as place field formation was shown to be affected by DB. Taken together, our results suggest that PF and DB are not mutually exclusive response properties of individual neurons. Rather, a cell's response mode depends on a variety of intrinsic properties, and modulation of these properties enables switching between PF and DB.

Activity-dependent mismatch between axo-axonic synapses and the axon initial segment controls neuronal output

The axon initial segment (AIS) is a structure at the start of the axon with a high density of sodium and potassium channels that defines the site of action potential generation. It has recently been shown that this structure is plastic and can change its position along the axon, as well as its length, in a homeostatic manner. Chronic activity-deprivation paradigms in a chick auditory nucleus lead to a lengthening of the AIS and an increase in neuronal excitability. On the other hand, a long-term increase in activity in dissociated rat hippocampal neurons results in an outward movement of the AIS and a decrease in the cell's excitability. Here, we investigated whether the AIS is capable of undergoing structural plasticity in rat hippocampal organotypic slices, which retain the diversity of neuronal cell types present at postnatal ages, including chandelier cells. These interneurons exclusively target the AIS of pyramidal neurons and form rows of presynaptic boutons along them. Stimulating individual CA1 pyramidal neurons that express channelrhodopsin-2 for 48 h leads to an outward shift of the AIS. Intriguingly, both the pre- and postsynaptic components of the axo-axonic synapses did not change position after AIS relocation. We used computational modeling to explore the functional consequences of this partial mismatch and found that it allows the GABAergic synapses to strongly oppose action potential generation, and thus downregulate pyramidal cell excitability. We propose that this spatial arrangement is the optimal configuration for a homeostatic response to long-term stimulation.

The Cell-Autonomous Role of Excitatory Synaptic Transmission in the Regulation of Neuronal Structure and Function

The cell-autonomous role of synaptic transmission in the regulation of neuronal structural and electrical properties is unclear. We have now employed a genetic approach to eliminate glutamatergic synaptic transmission onto individual CA1 pyramidal neurons in a mosaic fashion invivo. Surprisingly, while electrical properties are profoundly affected in these neurons, as well as inhibitory synaptic transmission, we found little perturbation of neuronal morphology, demonstrating a functional segregation of excitatory synaptic transmission from neuronal morphological development.

Homeostatic Plasticity: Single Hippocampal Neurons See the Light

Neurons adapt to altered network activity through homeostatic changes in synaptic function. In this issue of Neuron, Goold and Nicoll report that chronic hyperactivation of individual CA1 pyramidal neurons drives cell-autonomous, compensatory synapse elimination via CaMKIV-dependent transcription. These findings suggest that neurons gauge their intrinsic activity to instruct homeostatic regulation of synaptic inputs.

State-Dependent Dendritic Computation in Hippocampal CA1 Pyramidal Neurons

Depending on the behavioral state, hippocampal CA1 pyramidal neurons receive very distinct patterns of synaptic input and likewise produce very different output patterns. We have used simultaneous dendritic and somatic recordings and multisite glutamate uncaging to investigate the relationship between synaptic input pattern, the form of dendritic integration, and action potential output in CA1 neurons. We found that when synaptic input arrives asynchronously or highly distributed in space, the dendritic arbor performs a linear integration that allows the action potential rate and timing to vary as a function of the quantity of the input. In contrast, when synaptic input arrives synchronously and spatially clustered, the dendritic compartment receiving the clustered input produces a highly nonlinear integration that leads to an action potential output that is extraordinarily precise and invariant. We also present evidence that both of these forms of information processing may be independently engaged during the two distinct behavioral states of the hippocampus such that individual CA1 pyramidal neurons could perform two different state-dependent computations: input strength encoding during theta states and feature detection during sharp waves.

Development of spatial firing in the hippocampus of young rats.

The hippocampal formation participates in learning and memory, particularly that of a spatial nature. In adult rats, individual CA1 pyramidal neurons only fire when the animal visits specific locations in an environment, the "place field" of the neuron. Other structures (postsubiculum, thalamus, cingulum) contain neurons that code for the animal's instantaneous head direction. Previous work has shown that the rat hippocampal formation undergoes anatomical and neurophysiological maturation during the first 2 months of life and that rats <40 days of age are impaired in spatial navigation tasks. Here we show that the locational firing of CA1 pyramidal neurons is both less specific and less stable in animals aged <50 days. However, preliminary results indicate that head directional firing recorded around day 30 is essentially identical to that seen in adult animals. Therefore, the development of reliable, spatially specific place cell activity parallels the developmental time course of spatial navigational ability, but head directional firing appears before full maturation of the hippocampus.

Firing rates of hippocampal neurons are preserved during subsequent sleep episodes and modified by novel awake experience.

What determines the firing rate of cortical neurons in the absence of external sensory input or motor behavior, such as during sleep? Here we report that, in a familiar environment, the discharge frequency of simultaneously recorded individual CA1 pyramidal neurons and the coactivation of cell pairs remain highly correlated across sleep-wake-sleep sequences. However, both measures were affected when new sets of neurons were activated in a novel environment. Nevertheless, the grand mean firing rate of the whole pyramidal cell population remained constant across behavioral states and testing conditions. The findings suggest that long-term firing patterns of single cells can be modified by experience. We hypothesize that increased firing rates of recently used neurons are associated with a concomitant decrease in the discharge activity of the remaining population, leaving the mean excitability of the hippocampal network unaltered.

Na(+) dependence and the role of glutamate receptors and Na(+) channels in ion fluxes during hypoxia of rat hippocampal slices.

Spreading depression (SD) as well as hypoxia-induced SD-like depolarization in forebrain gray matter are characterized by near complete depolarization of neurons. The biophysical mechanism of the depolarization is not known. Earlier we reported that simultaneous pharmacological blockade of all known major Na(+) and Ca(2+) channels prevents hypoxic SD. We now recorded extracellular voltage, Na(+), and K(+) concentrations and the intracellular potential of individual CA1 pyramidal neurons during hypoxia of rat hippocampal tissue slices after substituting Na(+) in the bath by an impermeant cation, or in the presence of channel blocking drugs applied individually and in combination. Reducing extracellular Na(+) concentration [Na(+)](o) to 90 mM postponed the hypoxia-induced extracellular DC-potential deflection (DeltaV(o)) and reduced its amplitude, and it also postponed the SD-like depolarization of neurons. After lowering [Na(+)](o) to 25 mM, SD-like DeltaV(o) became very small, indicating that an influx of Na(+) is required for SD; influx of Ca(2+) ions alone is not sufficient. We then asked whether the SD-related Na(+) current flows through glutamate-controlled and/or through voltage-gated Na(+) channels. Administration of either the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), or the NMDA receptor antagonist (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) postponed the hypoxic DeltaV(o) and depressed its amplitude but the effect of the combined administration of these two drugs was not greater than that of either alone. During the early phase of hypoxia, before SD onset, [K(+)](o) increased faster and reached a much higher level in the presence of glutamate antagonists than in their absence. The [K(+)](o) level reached at the height of hypoxic SD was, however, not affected. When TTX was added to DNQX and CPP, SD was prevented in half the trials. When SD did occur, it was greatly delayed, yet eventually neurons depolarized to the same extent as in normal solution. The SD-related sudden drop in [Na(+)](o) was depressed by only 19% in the presence of the three drugs, indicating that Na(+) can flow into cells through pathways other than ionotropic glutamate receptors and TTX-sensitive Na(+) channels. We conclude that, when they are functional, glutamate-receptor-mediated and voltage-gated Na(+) currents are the major generators of the self-regenerative rapid depolarization, but in their absence other pathways can sometimes take their place. The final level of SD-like depolarization is determined by positive feedback and not by the number of channels available. A schematic flow chart of the events generating hypoxic SD is discussed.

Evidence for involvement of group II/III metabotropic glutamate receptors in NMDA receptor-independent long-term potentiation in area CA1 of rat hippocampus.

Previous studies implicated metabotropic glutamate receptors (mGluRs) in N-methyl-D-aspartate (NMDA) receptor-independent long-term potentiation (LTP) in area CA1 of the rat hippocampus. To learn more about the specific roles played by mGluRs in NMDA receptor-independent LTP, we used whole cell recordings to load individual CA1 pyramidal neurons with a G-protein inhibitor [guanosine-5'-O-(2-thiodiphosphate), GDPbetaS]. Although loading postsynaptic CA1 pyramidal neurons with GDPbetaS significantly reduced G-protein dependent postsynaptic potentials, GDPbetaS failed to prevent NMDA receptor- independent LTP, suggesting that postsynaptic G-protein-dependent mGluRs are not required. We also performed a series of extracellular field potential experiments in which we applied group-selective mGluR antagonists. We had previously determined that paired-pulse facilitation (PPF) was decreased during the first 30-45 min of NMDA receptor-independent LTP. To determine if mGluRs might be involved in these PPF changes, we used a twin-pulse stimulation protocol to measure PPF in field potential experiments. NMDA receptor-independent LTP was prevented by a group II mGluR antagonist [(2S)-alpha-ethylglutamic acid] and a group III mGluR antagonist [(RS)-alpha-cyclopropyl-4-phosphonophenylglycine], but was not prevented by other group II and III mGluR antagonists [(RS)-alpha-methylserine-O-phosphate monophenyl ester or (RS)-alpha-methylserine-O-phosphate]. NMDA receptor-independent LTP was not prevented by either of the group I mGluR antagonists we examined, (RS)-1-aminoindan-1,5-dicarboxylic acid and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester. The PPF changes which accompany NMDA receptor-independent LTP were not prevented by any of the group-selective mGluR antagonists we examined, even when the LTP itself was blocked. Finally, we found that tetanic stimulation in the presence of group III mGluR antagonists lead to nonspecific potentiation in control (nontetanized) input pathways. Taken together, our results argue against the involvement of postsynaptic group I mGluRs in NMDA receptor-independent LTP. Group II and/or group III mGluRs are required, but the specific details of the roles played by these mGluRs in NMDA receptor-independent LTP are uncertain. Based on the pattern of results we obtained, we suggest that group II mGluRs are required for induction of NMDA receptor-independent LTP, and that group III mGluRs are involved in determining the input specificity of NMDA receptor-independent LTP by suppressing potentiation of nearby, nontetanized synapses.

Global Ischemia Induces Downregulation of Glur2 mRNA and Increases AMPA Receptor-Mediated Ca2+Influx in Hippocampal CA1 Neurons of Gerbil

Transient, severe forebrain or global ischemia leads to delayed cell death of pyramidal neurons in the hippocampal CA1. The precise molecular mechanisms underlying neuronal cell death after global ischemia are as yet unknown. Glutamate receptor-mediated Ca2+ influx is thought to play a critical role in this cell death. In situ hybridization revealed that the expression of mRNA encoding GluR2 (the subunit that limits Ca2+ permeability of AMPA-type glutamate receptors) was markedly and specifically reduced in gerbil CA1 pyramidal neurons after global ischemia but before the onset of neurodegeneration. To determine whether the change in GluR2 expression is functionally significant, we examined the AMPA receptor-mediated rise in cytoplasmic free Ca2+ level ([Ca2+]i) in individual CA1 pyramidal neurons by optical imaging with the Ca2+ indicator dye fura-2 and by intracellular recording. Seventy-two hours after ischemia, CA1 neurons that retained the ability to fire action potentials exhibited a greatly enhanced AMPA-elicited rise in [Ca2+]i. Basal [Ca2+]i in these neurons was unchanged. These findings provide evidence for Ca2+ entry directly through AMPA receptors in pyramidal neurons destined to die. Downregulation of GluR2 gene expression and an increase in Ca2+ influx through AMPA receptors in response to endogenous glutamate are likely to contribute to the delayed neuronal death after global ischemia.

In vitro reinforcement of hippocampal bursting: a search for Skinner's atoms of behavior.

A novel "in vitro reinforcement" paradigm was used to investigate Skinner's (1953) hypotheses (a) that operant behavior is made up of infinitesimal "response elements" or "behavioral atoms" and (b) that these very small units, and not whole responses, are the functional units of reinforcement. Our tests are based on the assumption that behavioral atoms may plausibly be represented at the neural level by individual cellular responses. As a first approach, we attempted to reinforce the bursting responses of hippocampal units in a highly reduced brain-slice preparation with local micropressure applications of behaviorally reinforcing dopaminergic drugs. The same injections were administered independently of bursting to provide a "noncontingent" control for nonspecific stimulation or facilitation of firing. It was found that the bursting responses of individual CA1 pyramidal neurons may be progressively facilitated in a dose-related manner by response-contingent (but not noncontingent) injections of dopamine itself, the dopamine D1-preferring agonist SKF 82958, the D3-preferring agonist quinpirole, and the D2-like selective agonist (+)-4-propyl-9 hydroxynapthoxazine. These findings support the conclusion that unit bursting responses can be reinforced in vitro in hippocampal slices, and they further suggest that the same dopamine receptor subtypes are involved in both cellular and behavioral operant conditioning. The results thus provide indirect support for Skinner's atoms-of-behavior hypothesis.

A CELLULAR ANALOGUE OF OPERANT CONDITIONING

Using the hippocampal-slice preparation, we attempted to model operant conditioning in vitro by reinforcing pyramidal cell bursting responses with local micropressure applications of transmitters and drugs. The same injections were administered independently of bursting to provide a "noncontingent" control for direct pharmacological stimulation or facilitation of firing. The results suggested that the bursting responses of individual CA1 pyramidal neurons may be reinforced in a dose-related manner by response-contingent (but not noncontingent) injections of dopamine and the selective dopamine D2 agonist, N-0923. N-0924, a stereoisomer of N-0923 that is largely devoid of D2-agonist activity, failed to reinforce CA1 bursting. Burst-contingent injections of the excitatory neurotransmitter glutamate also failed to reinforce CA1 bursting; indeed, the glutamate applications (whether contingent or random) reduced the likelihood of bursts while increasing the frequency of solitary spikes. Reinforcement delays exceeding 200 ms largely eliminated the reinforcing efficacy of the D2 agonist N-0437 in CA1 operant conditioning. The results are consistent with the suggestion that the behaviorally reinforcing effects of dopaminergic agents can be modeled in vitro in the hippocampal-slice preparation.

Synaptic transmission in slices of rat hippocampus using a modified voltage clamp technique

A small modification to a voltage-clamp set-up for studying isolated neurons, and the use of simple hippocampal slices allowed stable recording of excitatory postsynaptic currents (EPSCs) that were evoked by stimulating the Shaffer's collaterals of individual CA1 pyramidal neurons. With the developed method EPSCs and focal extracellular potentials could be recorded simultaneously. It was confirmed that the EPSC consists of two components that are mediated via N-methyl-D-aspartate (NMDA)- and non-NMDA-receptors. The effects of different blockers of these receptors on the postsynaptic current were investigated, as were the effects of adenosine, which, depending on its concentration, could either depress or potentiate the synaptic transmission.

Cellular investigations of behavioral reinforcement

Using the hippocampal-slice preparation, we attempted to demonstrate operant conditioning of pyramidal cell activity using local micropressure applications of transmitters and drugs as reinforcement; the same injections administered independently of bursting provided a control for direct pharmacological stimulation or facilitation of firing. The results suggested that the spontaneous bursting of individual CA1 pyramidal neurons may be reinforced with activity-contingent injections of dopamine and cocaine, whereas, CA3-bursting responses may be reinforced with contingently-applied dynorphin A. We sought to confirm these indications of cellular reinforcement at the behavioral level in studies of hippocampal self-administration (despite the fact that the hippocampus has been ignored as a brain site for chemical self-administration experiments). The results suggested that dynorphin A is a powerful reinforcer of hippocampal self-administration behavior when injected in the CA3 field; experiments still in progress suggest that dopamine can reinforce self-administration behavior when injected in the CA1 field. Successful prediction of new behavioral data from operant-conditioning data at the cellular level helps to validate the cellular data by providing suggestive evidence of interrelationship between cellular and behavioral operant conditioning processes.