RESUMO
The ability of the mammalian brain to maintain spatial representations of external or internal information for short periods of time has been associated with sustained neuronal spiking and reverberatory neural network activity in the medial entorhinal cortex. Here, we show that conditional genetic deletion of netrin-1 or the netrin receptor deleted-in-colorectal cancer (DCC) from forebrain excitatory neurons leads to deficits in short-term spatial memory. We then demonstrate that conditional deletion of either netrin-1 or DCC inhibits cholinergic persistent firing and show that cholinergic activation of muscarinic receptors expressed by entorhinal cortical neurons promotes persistent firing by recruiting DCC to the plasma membrane. Together, these findings indicate that normal short-term spatial memory function requires the synergistic actions of acetylcholine and netrin-1.
Assuntos
Acetilcolina , Córtex Entorrinal , Animais , Acetilcolina/farmacologia , Netrina-1 , Prosencéfalo , Colinérgicos , MamíferosRESUMO
Long-term stable cell culture is a critical tool to better understand cell function. Most adherent cell culture models require a polymer substrate coating of poly-lysine or poly-ornithine for the cells to adhere and survive. However, polypeptide-based substrates are degraded by proteolysis and it remains a challenge to maintain healthy cell cultures for extended periods of time. Here, we report the development of an enhanced cell culture substrate based on a coating of dendritic polyglycerol amine (dPGA), a non-protein macromolecular biomimetic of poly-lysine, to promote the adhesion and survival of neurons in cell culture. We show that this new polymer coating provides enhanced survival, differentiation and long-term stability for cultures of primary neurons or neurons derived from human induced pluripotent stem cells (hiPSCs). Atomic force microscopy analysis provides evidence that greater nanoscale roughness contributes to the enhanced capacity of dPGA-coated surfaces to support cells in culture. We conclude that dPGA is a cytocompatible, functionally superior, easy to use, low cost and highly stable alternative to poly-cationic polymer cell culture substrate coatings such as poly-lysine and poly-ornithine. Summary statementHere, we describe a novel dendritic polyglycerol amine-based substrate coating, demonstrating superior performance compared to current polymer coatings for long-term culture of primary neurons and neurons derived from induced pluripotent stem cells.
Assuntos
Aminas , Células-Tronco Pluripotentes Induzidas , Técnicas de Cultura de Células , Diferenciação Celular , Glicerol , Humanos , Neurônios , PolímerosRESUMO
Adult neural plasticity engages mechanisms that change synapse structure and function, yet many of the underlying events bear a striking similarity to processes that occur during the initial establishment of neural circuits during development. It is a long-standing hypothesis that the molecular mechanisms critical for neural development may also regulate synaptic plasticity related to learning and memory in adults. Netrins were initially described as chemoattractant guidance cues that direct cell and axon migration during embryonic development, yet they continue to be expressed by neurons in the adult brain. Recent findings have identified roles for netrin-1 in synaptogenesis during postnatal maturation, and in synaptic plasticity in the adult mammalian brain, regulating AMPA glutamate receptor trafficking at excitatory synapses. These findings provide an example of a conserved developmental guidance cue that is expressed by neurons in the adult brain and functions as a key regulator of activity-dependent synaptic plasticity. Notably, in humans, genetic polymorphisms in netrin-1 and its receptors have been linked to neurodevelopmental and neurodegenerative disorders. The molecular mechanisms associated with the synaptic function of netrin-1 therefore present new therapeutic targets for neuropathologies associated with memory dysfunction. Here, we summarize recent findings that link netrin-1 signalling to synaptic plasticity, and discuss the implications of these discoveries for the neurobiological basis of memory consolidation.
Assuntos
Hipocampo , Plasticidade Neuronal , Animais , Encéfalo/metabolismo , Hipocampo/metabolismo , Humanos , Netrina-1/metabolismo , Sinapses/metabolismoRESUMO
The receptor deleted in colorectal cancer (DCC) and its ligand netrin-1 are essential for axon guidance during development and are expressed by neurons in the mature brain. Netrin-1 recruits GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and is critical for long-term potentiation (LTP) at CA3-CA1 hippocampal Schaffer collateral synapses, while conditional DCC deletion from glutamatergic neurons impairs hippocampal-dependent spatial memory and severely disrupts LTP induction. DCC co-fractionates with the detergent-resistant component of postsynaptic density, yet is enriched in axonal growth cones that differentiate into presynaptic terminals during development. Specific presynaptic and postsynaptic contributions of DCC to the function of mature neural circuits have yet to be identified. Employing hippocampal subregion-specific conditional deletion of DCC, we show that DCC loss from CA1 hippocampal pyramidal neurons resulted in deficits in spatial memory, increased resting membrane potential, abnormal dendritic spine morphology, weaker spontaneous excitatory postsynaptic activity, and reduced levels of postsynaptic adaptor and signaling proteins; however, the capacity to induce LTP remained intact. In contrast, deletion of DCC from CA3 neurons did not induce detectable changes in the intrinsic electrophysiological properties of CA1 pyramidal neurons, but impaired performance on the novel object place recognition task as well as compromised excitatory synaptic transmission and LTP at Schaffer collateral synapses. Together, these findings reveal specific pre- and post-synaptic contributions of DCC to hippocampal synaptic plasticity underlying spatial memory.
Assuntos
Envelhecimento/metabolismo , Receptor DCC/metabolismo , Hipocampo/metabolismo , Consolidação da Memória , Sinapses/metabolismo , Animais , Região CA1 Hipocampal/metabolismo , Região CA3 Hipocampal/metabolismo , Espinhas Dendríticas/metabolismo , Deleção de Genes , Ácido Glutâmico , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Células Piramidais/metabolismo , Memória EspacialRESUMO
The numbers and strengths of synapses in the brain change throughout development, and even into adulthood, as synaptic inputs are added, eliminated, and refined in response to ongoing neural activity. A number of experimental techniques can assess these changes, including single-cell electrophysiological recording which offers measurements of synaptic inputs with high temporal resolution. Coupled with electrical stimulation, photoactivatable opsins, and caged compounds, to facilitate fine spatiotemporal control over release of neurotransmitters, electrophysiological recordings allow for precise dissection of presynaptic and postsynaptic mechanisms of action. Here, we discuss the strengths and pitfalls of various techniques commonly used to analyze synapses, including miniature excitatory/inhibitory (E/I) postsynaptic currents, evoked release, and optogenetic stimulation. Together, these techniques can provide multiple lines of convergent evidence to generate meaningful insight into the emergence of circuit connectivity and maturation. A full understanding of potential caveats and alternative explanations for findings is essential to avoid data misinterpretation.
RESUMO
Netrin-1 was initially characterized as an axon guidance molecule that is essential for normal embryonic neural development; however, many types of neurons continue to express netrin-1 in the postnatal and adult mammalian brain. Netrin-1 and the netrin receptor DCC are both enriched at synapses. In the adult hippocampus, activity-dependent secretion of netrin-1 by neurons potentiates glutamatergic synapse function, and is critical for long-term potentiation, an experimental cellular model of learning and memory. Here, we assessed the impact of neuronal expression of netrin-1 in the adult brain on behavior using tests of learning and memory. We show that adult mice exhibit impaired spatial memory following conditional deletion of netrin-1 from glutamatergic neurons in the hippocampus and neocortex. Further, we provide evidence that mice with conditional deletion of netrin-1 do not display aberrant anxiety-like phenotypes and show a reduction in self-grooming behavior. These findings reveal a critical role for netrin-1 expressed by neurons in the regulation of spatial memory formation.
Assuntos
Hipocampo/fisiologia , Neocórtex/fisiologia , Netrina-1/fisiologia , Neurônios/fisiologia , Memória Espacial/fisiologia , Animais , Comportamento Animal , Feminino , Ácido Glutâmico/fisiologia , Hipocampo/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neocórtex/metabolismo , Netrina-1/metabolismo , Neurônios/metabolismoRESUMO
Dynamic trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPARs) to synapses is critical for activity-dependent synaptic plasticity underlying learning and memory, but the identity of key molecular effectors remains elusive. Here, we demonstrate that membrane depolarization and N-methyl-D-aspartate receptor (NMDAR) activation triggers secretion of the chemotropic guidance cue netrin-1 from dendrites. Using selective genetic deletion, we show that netrin-1 expression by excitatory neurons is required for NMDAR-dependent long-term potentiation (LTP) in the adult hippocampus. Furthermore, we demonstrate that application of exogenous netrin-1 is sufficient to trigger the potentiation of excitatory glutamatergic transmission at hippocampal Schaffer collateral synapses via Ca2+-dependent recruitment of GluA1-containing AMPARs, promoting the maturation of immature or nascent synapses. These findings identify a central role for activity-dependent release of netrin-1 as a critical effector of synaptic plasticity in the adult hippocampus.
Assuntos
Hipocampo/metabolismo , Netrina-1/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Potenciação de Longa Duração/fisiologia , Camundongos , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Rapid eye movement sleep (REMS) has been linked with spatial and emotional memory consolidation. However, establishing direct causality between neural activity during REMS and memory consolidation has proven difficult because of the transient nature of REMS and significant caveats associated with REMS deprivation techniques. In mice, we optogenetically silenced medial septum γ-aminobutyric acid-releasing (MS(GABA)) neurons, allowing for temporally precise attenuation of the memory-associated theta rhythm during REMS without disturbing sleeping behavior. REMS-specific optogenetic silencing of MS(GABA) neurons selectively during a REMS critical window after learning erased subsequent novel object place recognition and impaired fear-conditioned contextual memory. Silencing MS(GABA) neurons for similar durations outside REMS episodes had no effect on memory. These results demonstrate that MS(GABA) neuronal activity specifically during REMS is required for normal memory consolidation.
Assuntos
Neurônios GABAérgicos/fisiologia , Consolidação da Memória/fisiologia , Sono REM/fisiologia , Ritmo Teta/fisiologia , Animais , Região CA1 Hipocampal/fisiologia , Condicionamento Psicológico/fisiologia , Medo/fisiologia , Inativação Gênica , Aprendizagem/fisiologia , Camundongos , Camundongos Transgênicos , Optogenética , Privação do Sono/fisiopatologia , Sono REM/genética , Ritmo Teta/genética , Vigília/fisiologiaRESUMO
Netrin-1 is a secreted protein that directs long-range axon guidance during early stages of neural circuit formation and continues to be expressed in the mammalian forebrain during the postnatal period of peak synapse formation. Here we demonstrate a synaptogenic function of netrin-1 in rat and mouse cortical neurons and investigate the underlying mechanism. We report that netrin-1 and its receptor DCC are widely expressed by neurons in the developing mammalian cortex during synapse formation and are enriched at synapses in vivo. We detect DCC protein distributed along the axons and dendrites of cultured cortical neurons and provide evidence that newly translated netrin-1 is selectively transported to dendrites. Using gain and loss of function manipulations, we demonstrate that netrin-1 increases the number and strength of excitatory synapses made between developing cortical neurons. We show that netrin-1 increases the complexity of axon and dendrite arbors, thereby increasing the probability of contact. At sites of contact, netrin-1 promotes adhesion, while locally enriching and reorganizing the underlying actin cytoskeleton through Src family kinase signaling and m-Tor-dependent protein translation to locally cluster presynaptic and postsynaptic proteins. Finally, we demonstrate using whole-cell patch-clamp electrophysiology that netrin-1 increases the frequency and amplitude of mEPSCs recorded from cortical pyramidal neurons. These findings identify netrin-1 as a synapse-enriched protein that promotes synaptogenesis between mammalian cortical neurons.
Assuntos
Córtex Cerebral/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Fatores de Crescimento Neural/fisiologia , Células Piramidais/metabolismo , Sinapses/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Crescimento Neural/biossíntese , Netrina-1 , Neurogênese/genética , Células Piramidais/fisiologia , Ratos , Ratos Sprague-Dawley , Sinapses/fisiologia , Proteínas Supressoras de Tumor/biossínteseRESUMO
Rapid-eye movement (REM) sleep correlates with neuronal activity in the brainstem, basal forebrain and lateral hypothalamus. Lateral hypothalamus melanin-concentrating hormone (MCH)-expressing neurons are active during sleep, but their effects on REM sleep remain unclear. Using optogenetic tools in newly generated Tg(Pmch-cre) mice, we found that acute activation of MCH neurons (ChETA, SSFO) at the onset of REM sleep extended the duration of REM, but not non-REM, sleep episodes. In contrast, their acute silencing (eNpHR3.0, archaerhodopsin) reduced the frequency and amplitude of hippocampal theta rhythm without affecting REM sleep duration. In vitro activation of MCH neuron terminals induced GABAA-mediated inhibitory postsynaptic currents in wake-promoting histaminergic neurons of the tuberomammillary nucleus (TMN), and in vivo activation of MCH neuron terminals in TMN or medial septum also prolonged REM sleep episodes. Collectively, these results suggest that activation of MCH neurons maintains REM sleep, possibly through inhibition of arousal circuits in the mammalian brain.
Assuntos
Hipotálamo/fisiologia , Rede Nervosa/fisiologia , Vias Neurais/fisiologia , Optogenética , Sono REM/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Animais Recém-Nascidos , Bicuculina/farmacologia , Channelrhodopsins , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Regulação da Expressão Gênica , Hormônios Hipotalâmicos/genética , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Melaninas/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rede Nervosa/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Hormônios Hipofisários/genética , Ritmo Teta/efeitos dos fármacos , Ritmo Teta/genética , Transdução Genética , Valina/análogos & derivados , Valina/farmacologiaRESUMO
The parasubiculum (PaS) is a component of the hippocampal formation that sends its major output to layer II of the entorhinal cortex. The PaS receives strong cholinergic innervation from the basal forebrain that is likely to modulate neuronal excitability and contribute to theta-frequency network activity. The present study used whole cell current- and voltage-clamp recordings to determine the effects of cholinergic receptor activation on layer II PaS neurons. Bath application of carbachol (CCh; 10-50 µM) resulted in a dose-dependent depolarization of morphologically-identified layer II stellate and pyramidal cells that was not prevented by blockade of excitatory and inhibitory synaptic inputs. Bath application of the M1 receptor antagonist pirenzepine (1 µM), but not the M2-preferring antagonist methoctramine (1 µM), blocked the depolarization, suggesting that it is dependent on M1 receptors. Voltage-clamp experiments using ramped voltage commands showed that CCh resulted in the gradual development of an inward current that was partially blocked by concurrent application of the selective Kv7.2/3 channel antagonist XE-991, which inhibits the muscarine-dependent K(+) current I M. The remaining inward current also reversed near EK and was inhibited by the K(+) channel blocker Ba(2+), suggesting that M1 receptor activation attenuates both I M as well as an additional K(+) current. The additional K(+) current showed rectification at depolarized voltages, similar to K(+) conductances mediated by Kir 2.3 channels. The cholinergic depolarization of layer II PaS neurons therefore appears to occur through M1-mediated effects on I M as well as an additional K(+) conductance.
Assuntos
Potenciais da Membrana/fisiologia , Neurônios/metabolismo , Células Piramidais/metabolismo , Receptor Muscarínico M1/metabolismo , Animais , Antracenos/farmacologia , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Diaminas/farmacologia , Canal de Potássio KCNQ2/antagonistas & inibidores , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/antagonistas & inibidores , Canal de Potássio KCNQ3/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Antagonistas Muscarínicos/farmacologia , Neurônios/citologia , Parassimpatolíticos/farmacologia , Pirenzepina/farmacologia , Células Piramidais/citologia , Ratos , Ratos Long-Evans , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inibidoresRESUMO
The transmembrane protein deleted in colorectal cancer (DCC) and its ligand, netrin-1, regulate synaptogenesis during development, but their function in the mature central nervous system is unknown. Given that DCC promotes cell-cell adhesion, is expressed by neurons, and activates proteins that signal at synapses, we hypothesized that DCC expression by neurons regulates synaptic function and plasticity in the adult brain. We report that DCC is enriched in dendritic spines of pyramidal neurons in wild-type mice, and we demonstrate that selective deletion of DCC from neurons in the adult forebrain results in the loss of long-term potentiation (LTP), intact long-term depression, shorter dendritic spines, and impaired spatial and recognition memory. LTP induction requires Src activation of NMDA receptor (NMDAR) function. DCC deletion severely reduced Src activation. We demonstrate that enhancing NMDAR function or activating Src rescues LTP in the absence of DCC. We conclude that DCC activation of Src is required for NMDAR-dependent LTP and certain forms of learning and memory.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Receptores de Superfície Celular/metabolismo , Sinapses/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Envelhecimento/metabolismo , Animais , Receptor DCC , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Ativação Enzimática , Deleção de Genes , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/fisiopatologia , Potenciação de Longa Duração , Aprendizagem em Labirinto , Memória , Camundongos , Fatores de Crescimento Neural/metabolismo , Netrina-1 , Neurônios/patologia , Neurônios/ultraestrutura , Fosfolipase C gama/metabolismo , Fosforilação , Prosencéfalo/metabolismo , Prosencéfalo/patologia , Prosencéfalo/fisiopatologia , Ratos , Receptores de Superfície Celular/deficiência , Receptores de N-Metil-D-Aspartato/metabolismo , Frações Subcelulares/metabolismo , Sinapses/patologia , Sinapses/ultraestrutura , Proteínas Supressoras de Tumor/deficiência , Quinases da Família src/metabolismoRESUMO
Ionic conductances that generate membrane potential oscillations in neurons of layer II of the parasubiculum were studied using whole cell current-clamp recordings in horizontal slices from the rat brain. Blockade of ionotropic glutamate and GABA synaptic transmission did not reduce the power of the oscillations, indicating that oscillations are not dependent on synaptic inputs. Oscillations were eliminated when cells were hyperpolarized 6-10 mV below spike threshold, indicating that they are mediated by voltage-dependent conductances. Application of TTX completely eliminated oscillations, suggesting that Na(+) currents are required for the generation of the oscillations. Oscillations were not reduced by blocking Ca(2+) currents with Cd(2+) or Ca(2+)-free artificial cerebrospinal fluid, or by blocking K(+) conductances with either 50 microM or 5 mM 4-aminopyridine (4-AP), 30 mM tetraethylammonium (TEA), or Ba(2+)(1-2 mM). Oscillations also persisted during blockade of the muscarinic-dependent K(+) current, I(M), using the selective antagonist XE-991 (10 microM). However, oscillations were significantly attenuated by blocking the hyperpolarization-activated cationic current I(h) with Cs(+) and were almost completely blocked by the more potent I(h) blocker ZD7288 (100 microM). Intrinsic membrane potential oscillations in neurons of layer II of the parasubiculum are therefore likely driven by an interaction between an inward persistent Na(+) current and time-dependent deactivation of I(h). These voltage-dependent conductances provide a mechanism for the generation of membrane potential oscillations that can help support rhythmic network activity within the parasubiculum during theta-related behaviors.
Assuntos
Relógios Biológicos/fisiologia , Hipocampo/citologia , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Ritmo Teta , 4-Aminopiridina/farmacologia , Animais , Bicuculina/farmacologia , Relógios Biológicos/efeitos dos fármacos , Relógios Biológicos/efeitos da radiação , Bloqueadores dos Canais de Cálcio/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Técnicas In Vitro , Ácido Cinurênico/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos da radiação , Técnicas de Patch-Clamp/métodos , Ácidos Fosfínicos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Propanolaminas/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Long-Evans , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Ritmo Teta/efeitos dos fármacosRESUMO
The entorhinal cortex receives a large projection from the piriform cortex, and synaptic plasticity in this pathway may affect olfactory processing. In vitro whole cell recordings have been used here to investigate postsynaptic signalling mechanisms that mediate the induction of long-term synaptic depression (LTD) in layer II entorhinal cortex cells. To induce LTD, pairs of pulses, using a 30-millisecond interval, were delivered at 1 Hz for 15 minutes. Induction of LTD was blocked by the NMDA receptor antagonist APV and by the calcium chelator BAPTA, consistent with a requirement for calcium influx via NMDA receptors. Induction of LTD was blocked when the FK506 was included in the intracellular solution to block the phosphatase calcineurin. Okadaic acid, which blocks activation of protein phosphatases 1 and 2a, also prevented LTD. Activation of protein phosphatases following calcium influx therefore contributes to induction of LTD in layer II of the entorhinal cortex.
Assuntos
Córtex Entorrinal/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Neurônios/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Calcineurina/fisiologia , Sinalização do Cálcio , Ciclosporina/farmacologia , Maleato de Dizocilpina/farmacologia , Córtex Entorrinal/citologia , Córtex Entorrinal/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Masculino , Neurônios/efeitos dos fármacos , Ácido Okadáico/farmacologia , Técnicas de Patch-Clamp , Proteína Fosfatase 1/fisiologia , Células Piramidais/fisiologia , Ratos , Ratos Long-Evans , Receptores de N-Metil-D-Aspartato/fisiologia , Tacrolimo/farmacologiaRESUMO
The parasubiculum is a major component of the hippocampal formation that receives inputs from the CA1 region, anterior thalamus, and medial septum and that projects primarily to layer II of the entorhinal cortex. Hippocampal theta-frequency (4-12 Hz) electroencephalographic (EEG) activity has been correlated with sensorimotor integration, spatial navigation, and memory functions. The present study was aimed at determining if theta is also generated locally within the parasubiculum versus volume conducted from adjacent structures. In urethan-anesthetized rats, the phase-reversal of theta activity between superficial and deep layers of the parasubiculum was demonstrated using differential recordings from movable bipolar electrodes that eliminate the influence of volume-conducted activity. Parasubicular theta was abolished by atropine, and was in phase with theta in stratum radiatum/lacunosum-moleculare of the CA1 region. Whole cell current-clamp recordings in brain slices were then used to determine if parasubicular theta may be generated in part by membrane potential oscillations in layer II neurons. Membrane potential oscillations occurred in most layer II neurons, including four putative interneurons, when cells were held at near-threshold voltages using current injection. The frequency of oscillations increased from 3.2 to 6.1 Hz when bath temperature was raised from 22 to 32 degrees C, and oscillations persisted in the presence of blockers of fast ionotropic glutamatergic and GABAergic synaptic transmission. Oscillations are therefore likely generated by intrinsic, voltage-dependent ionic conductances. These results indicate that theta field activity is generated locally within the parasubiculum and that intrinsic membrane potential oscillations, synchronized by local inhibitory inputs, may contribute to the generation of this activity.