Optogenetics with microbial opsin genes, and pharmacogenetics with designer receptors, represent potent and versatile experimental modalities that can be integrated with each other as well as with a rich diversity of synergistic methods to provide fundamental opportunities in neuroscience research. Since initial steps were taken with these approaches less than 10 years ago, we are witnessing a rapid rise in publications (Fig. 1). The 7th Annual Brain Research Meeting in New Orleans in October 2012, Optogenetics and Pharmacogenetics in Neuronal Function and Dysfunction, brought together leading researchers that have developed and used these tools to explore a wide range of questions in nervous system function and dysfunction. This special issue of Brain Research includes articles by speakers in this meeting and others, which together synthesize and summarize the state of the art for optogenetics and designer receptors.
Optogenetics; Pharmacogenetics; Channelrhodopsin; DREADD; Designer receptor; Viral transduction; Neurotransmitter signalling; Motivation/cognition; Autonomic function; Sensory Function; Translational neuroscience
The effect of electrical stimulation on neuronal membrane potential is frequency dependent. Low frequency electrical stimulation can evoke action potentials, whereas high frequency stimulation can inhibit action potential transmission. Optical stimulation of channelrhodopsin-2 (ChR2) expressed in neuronal membranes can also excite action potentials. However, it is unknown whether optical stimulation of ChR2-expressing neurons produces a transition from excitation to inhibition with increasing light pulse frequencies. Here we report optical inhibition of motor neuron and muscle activity in vivo in the cooled sciatic nerves of Thy1-ChR2-EYFP mice. We also demonstrate all-optical single-wavelength control of neuronal excitation and inhibition without co-expression of inhibitory and excitatory opsins. This all-optical system is free from stimulation-induced electrical artifacts and thus provides a new approach to investigate mechanisms of high frequency inhibition in neuronal circuits in vivo and in vitro.
In Huntington’s disease (HD) mouse models, spontaneous inhibitory synaptic activity is enhanced in a subpopulation of medium-sized spiny neurons (MSNs), which could dampen striatal output. We examined the potential source(s) of increased inhibition using electrophysiological and optogenetic methods to assess feedback and feedforward inhibition in two transgenic mouse models of HD. Single whole-cell patch-clamp recordings demonstrated that increased GABA synaptic activity impinges principally on indirect pathway MSNs. Dual patch recordings between MSNs demonstrated reduced connectivity between MSNs in HD mice. However, while connectivity was strictly unidirectional in controls, in HD mice bidirectional connectivity occurred. Other sources of increased GABA activity in MSNs also were identified. Dual patch recordings from fast spiking (FS) interneuron–MSN pairs demonstrated greater but variable amplitude responses in MSNs. In agreement, selective optogenetic stimulation of parvalbumin-expressing, FS interneurons induced significantly larger amplitude MSN responses in HD compared with control mice. While there were no differences in responses of MSNs evoked by activating single persistent low-threshold spiking (PLTS) interneurons in recorded pairs, these interneurons fired more action potentials in both HD models, providing another source for increased frequency of spontaneous GABA synaptic activity in MSNs. Selective optogenetic stimulation of somatostatin-expressing, PLTS interneurons did not reveal any significant differences in responses of MSNs in HD mice. These findings provide strong evidence that both feedforward and to a lesser extent feedback inhibition to MSNs in HD can potentially be sources for the increased GABA synaptic activity of indirect pathway MSNs.
The basolateral amygdala (BLA) plays a crucial role in emotional learning irrespective of valence1–5. While the BLA projection to the nucleus accumbens (NAc) is hypothesized to modulate cue-triggered motivated behaviors4, 6, 7,, our understanding of the interaction between these two brain regions has been limited by the inability to manipulate neural circuit elements of this pathway selectively during behavior. To circumvent this limitation, we used in vivo optogenetic stimulation or inhibition of glutamatergic fibers from the BLA to the NAc, coupled with intracranial pharmacology and ex vivo electrophysiology. We show that optical stimulation of the BLA-to-NAc pathway in mice reinforces behavioral responding to earn additional optical stimulations of these synaptic inputs. Optical stimulation of BLA-to-NAc glutamatergic fibers required intra-NAc dopamine D1-type, but not D2-type, receptor signaling. Brief optical inhibition of BLA-to-NAc fibers reduced cue-evoked intake of sucrose, demonstrating an important role of this specific pathway in controlling naturally occurring reward-related behavior. Moreover, while optical stimulation of medial prefrontal cortex (mPFC) to NAc glutamatergic fibers also elicited reliable excitatory synaptic responses, optical self-stimulation behavior was not observed by activation of this pathway. These data suggest that while the BLA is important for processing both positive and negative affect, the BLA-to-NAc glutamatergic pathway in conjunction with dopamine signaling in the NAc promotes motivated behavioral responding.
Postsynaptic spines at CA3-CA1 synapses differ in glutamate receptor composition according to the hemispheric origin of CA3 afferents. To study the resulting functional consequences, we used optogenetic tools to selectively stimulate axons of CA3 pyramidal cells originating in either left or right mouse hippocampus. We found that left CA3 input produced more long-term potentiation at CA1 synapses than right CA3 input, due to differential expression of GluN2B subunit-containing NMDA receptors.
STDP; LTP; Channelrhodopsin-2; hippocampus; mouse
Optogenetic control of the peripheral nervous system (PNS) would enable novel studies of motor control, somatosensory transduction, and pain processing. Such control requires the development of methods to deliver opsins and light to targeted sub-populations of neurons within peripheral nerves. We report here methods to deliver opsins and light to targeted peripheral neurons and robust optogenetic modulation of motor neuron activity in freely moving, non-transgenic mammals. We show that intramuscular injection of adeno-associated virus serotype 6 enables expression of channelrhodopsin (ChR2) in motor neurons innervating the injected muscle. Illumination of nerves containing mixed populations of axons from these targeted neurons and from neurons innervating other muscles produces ChR2-mediated optogenetic activation restricted to the injected muscle. We demonstrate that an implanted optical nerve cuff is well-tolerated, delivers light to the sciatic nerve, and optically stimulates muscle in freely moving rats. These methods can be broadly applied to study PNS disorders and lay the groundwork for future therapeutic application of optogenetics.
The C1 neurons are a nodal point for blood pressure control and other autonomic responses. Here we test whether these rostral ventrolateral medullary catecholaminergic (RVLM-CA) neurons use glutamate as a transmitter in the dorsal motor nucleus of the vagus (DMV). After injecting Cre-dependent AAV2 DIO-Ef1α-channelrhodopsin2(ChR2)-mCherry (AAV2) into the RVLM of dopamine-beta-hydroxylase Cre transgenic mice (DβHCre/0), mCherry was detected exclusively in RVLM-CA neurons. Within the DMV >95% mCherry-immunoreactive (-ir) axonal varicosities were tyrosine hydroxylase-ir and the same proportion were vesicular glutamate transporter 2 (VGLUT2)-ir. VGLUT2-mCherry co-localization was virtually absent when AAV2 was injected into the RVLM of DβHCre/0;VGLUT2flox/flox mice, into the caudal VLM (A1 noradrenergic neuron-rich region) of DβHCre/0 mice or into the raphe of ePetCre/0 mice. Following injection of AAV2 into RVLM of TH-Cre rats, phenylethanolamine N-methyl transferase (PNMT) and VGLUT2 immunoreactivities were highly co-localized in DMV within EYFP-positive or EYFP-negative axonal varicosities. Ultrastructurally, mCherry terminals from RVLM-CA neurons in DβHCre/0 mice made predominantly asymmetric synapses with ChAT-ir DMV neurons. Photostimulation of ChR2-positive axons in DβHCre/0 mouse brain slices produced EPSCs in 71% of tested DMV preganglionic neurons (PGNs) but no IPSCs. Photostimulation (20 Hz) activated PGNs up to 8 spikes/sec (current clamp). EPSCs were eliminated by tetrodotoxin, reinstated by 4-aminopyridine and blocked by ionotropic glutamate receptor blockers. In conclusion, VGLUT2 is expressed by RVLM-CA (C1) neurons in rats and mice regardless of the presence of AAV2, the C1 neurons activate DMV parasympathetic preganglionic neurons monosynaptically and this connection uses glutamate as an ionotropic transmitter.
Neuroscientists have made impressive advances in understanding the microscale function of single neurons and the macroscale activity of the human brain. One can probe molecular and biophysical aspects of individual neurons and also view the human brain in action with magnetic resonance imaging (MRI) or magnetoencephalography (MEG). However, the mechanisms of perception, cognition, and action remain mysterious because they emerge from the real-time interactions of large sets of neurons in densely interconnected, widespread neural circuits.
Ventral tegmental area (VTA) dopamine (DA) neurons in the brain’s reward circuit play a crucial role in mediating stress responses1–4 including determining susceptibility vs. resilience to social stress-induced behavioural abnormalities5. VTA DA neurons exhibit two in vivo patterns of firing: low frequency tonic firing and high frequency phasic firing6–8. Phasic firing of the neurons, which is well known to encode reward signals6,7,9, is upregulated by repeated social defeat stress, a highly validated mouse model of depression5,8,10–13. Surprisingly, this pathophysiological effect is seen in susceptible mice only, with no change in firing rate apparent in resilient individuals5,8. However, direct evidence linking—in real-time—DA neuron phasic firing in promoting the susceptible (depression-like) phenotype is lacking. Here, we took advantage of the temporal precision and cell type- and projection pathway-specificity of optogenetics to demonstrate that enhanced phasic firing of these neurons mediates susceptibility to social defeat stress in freely behaving mice. We show that optogenetic induction of phasic, but not tonic, firing, in VTA DA neurons of mice undergoing a subthreshold social defeat paradigm rapidly induced a susceptible phenotype as measured by social avoidance and decreased sucrose preference. Optogenetic phasic stimulation of these neurons also quickly induced a susceptible phenotype in previously resilient mice that had been subjected to repeated social defeat stress. Furthermore, we show differences in projection pathway-specificity in promoting stress susceptibility: phasic activation of VTA neurons projecting to the nucleus accumbens (NAc), but not to the medial prefrontal cortex (mPFC), induced susceptibility to social defeat stress. Conversely, optogenetic inhibition of the VTA-NAc projection induced resilience, while inhibition of the VTA-mPFC projection promoted susceptibility. Overall, these studies reveal novel firing pattern- and neural circuit-specific mechanisms of depression.
Relapse to maladaptive eating habits during dieting is often provoked by stress. Recently, we identified a role of dorsal medial prefrontal cortex (mPFC) neurons in stress-induced reinstatement of palatable food seeking in male rats. It is unknown whether endogenous neural activity in dorsal mPFC drives stress-induced reinstatement in female rats. Here, we used an optogenetic approach, in which female rats received bilateral dorsal mPFC microinjections of viral constructs coding light-sensitive eNpHR3.0 – eYFP or control eYFP protein and intracranial fiber optic implants. Rats were food restricted and trained to lever press for palatable food pellets. Subsequently, pellets were removed, and lever pressing was extinguished; then the effect of bilateral dorsal mPFC light delivery on reinstatement of food seeking was assessed after injections of the pharmacological stressor yohimbine (an α-2 andrenoceptor antagonist) or pellet priming, a manipulation known to provoke food seeking in hungry rats. Dorsal mPFC light delivery attenuated yohimbine-induced reinstatement of food seeking in eNpHR3.0-injected but not eYFP-injected rats. This optical manipulation had no effect on pellet-priming-induced reinstatement or ongoing food-reinforced responding. Dorsal mPFC light delivery attenuated yohimbine-induced Fos immuno-reactivity and disrupted neural activity during in vivo electrophysiological recording in awake rats. Optical stimulation caused significant outward currents and blocked electrically evoked action potentials in eNpHR3.0-injected but not eYFP-injected mPFC hemispheres. Light delivery alone caused no significant inflammatory response in mPFC. These findings indicate that intracranial light delivery in eNpHR3.0 rats disrupts endogenous dorsal mPFC neural activity that plays a role in stress-induced relapse to food seeking in female rats.
Cerebrocortical injuries, such as stroke, are a major source of disability. Maladaptive consequences can result from post-injury local reorganization of cortical circuits. For example, epilepsy is a common sequela of cortical stroke, yet mechanisms responsible for seizures following cortical injuries remain unknown. In addition to local reorganization, long-range, extra-cortical connections might be critical for seizure maintenance. Here we report in rats the first evidence that the thalamus – a structure remote from but connected to the injured cortex – is required to maintain cortical seizures. Thalamocortical neurons connected to the injured epileptic cortex undergo changes in HCN channel expression and become hyperexcitable. Targeting these neurons with a closed-loop optogenetic strategy demonstrates that reducing their activity in real-time is sufficient to immediately interrupt electrographic and behavioral seizures. This approach is of therapeutic interest for intractable epilepsy, since it spares cortical function between seizures, in contrast to existing treatments such as surgical lesioning or drugs.
We demonstrate a two-photon optogenetic method that generates action potentials in neurons with single-cell precision, using the red-shifted opsin C1V1T. We apply the method to optically map synaptic circuits in mouse neocortical brain slices and to activate small dendritic regions and individual spines. Using a spatial light modulator we split the laser beam onto several neurons and perform simultaneous optogenetic activation of selected neurons in three dimensions.
Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function.
Neocortical epilepsy is frequently drug-resistant. Surgery to remove the epileptogenic zone is only feasible in a minority of cases, leaving many patients without an effective treatment. We report the potential efficacy of gene therapy in focal neocortical epilepsy using a rodent model in which epilepsy is induced by tetanus toxin injection in the motor cortex. By applying several complementary methods that use continuous wireless electroencephalographic monitoring to quantify epileptic activity, we observed increases in high frequency activity and in the occurrence of epileptiform events. Pyramidal neurons in the epileptic focus showed enhanced intrinsic excitability consistent with seizure generation. Optogenetic inhibition of a subset of principal neurons transduced with halorhodopsin targeted to the epileptic focus by lentiviral delivery was sufficient to attenuate electroencephalographic seizures. Local lentiviral overexpression of the potassium channel Kv1.1 reduced the intrinsic excitability of transduced pyramidal neurons. Coinjection of this Kv1.1 lentivirus with tetanus toxin fully prevented the occurrence of electroencephalographic seizures. Finally, administration of the Kv1.1 lentivirus to an established epileptic focus progressively suppressed epileptic activity over several weeks without detectable behavioral side effects. Thus, gene therapy in a rodent model can be used to suppress seizures acutely, prevent their occurrence after an epileptogenic stimulus, and successfully treat established focal epilepsy.
Cortical gamma oscillations (20–80 Hz) predict increases in focused attention, and failure in gamma regulation is a hallmark of neurological and psychiatric disease. Current theory predicts that gamma oscillations are generated by synchronous activity of fast-spiking inhibitory interneurons, with the resulting rhythmic inhibition producing neural ensemble synchrony by generating a narrow window for effective excitation. We causally tested these hypotheses in barrel cortex in vivo by targeting optogenetic manipulation selectively to fast-spiking interneurons. Here we show that light-driven activation of fast-spiking interneurons at varied frequencies (8–200 Hz) selectively amplifies gamma oscillations. In contrast, pyramidal neuron activation amplifies only lower frequency oscillations, a cell-type-specific double dissociation. We found that the timing of a sensory input relative to a gamma cycle determined the amplitude and precision of evoked responses. Our data directly support the fast-spiking-gamma hypothesis and provide the first causal evidence that distinct network activity states can be induced in vivo by cell-type-specific activation.
A major long-term goal of systems neuroscience is to identify the different roles of neural subtypes in brain circuit function. The ability to causally manipulate selective cell types is critical to meeting this goal. This protocol describes techniques for optically stimulating specific populations of excitatory neurons and inhibitory interneurons in vivo in combination with electrophysiology. Cell type selectivity is obtained using Cre-dependent expression of the light-activated channel Channelrhodopsin-2. We also describe approaches for minimizing optical interference with simultaneous extracellular and intracellular recording. These optogenetic techniques provide a spatially and temporally precise means of studying neural activity in the intact brain and allow a detailed examination of the effect of evoked activity on the surrounding local neural network. Injection of viral vectors requires 30–45 min, and in vivo electrophysiology with optogenetic stimulation requires 1–4 h.
Channelrhodopsin-2 (ChR2) is a light-gated, cation-selective ion channel isolated from the green algae Chlamydomonas reinhardtii. Here, we report the generation of transgenic mice that express a ChR2-YFP fusion protein in the CNS for in vivo activation and mapping of neural circuits. Using focal illumination of the cerebral cortex and olfactory bulb, we demonstrate a highly reproducible, light-dependent activation of neurons and precise control of firing frequency in vivo. To test the feasibility of mapping neural circuits, we exploited the circuitry formed between the olfactory bulb and the piriform cortex in anesthetized mice. In the olfactory bulb, individual mitral cells fired action potentials in response to light, and their firing rate was not influenced by costimulated glomeruli. However, in piriform cortex, the activity of target neurons increased as larger areas of the bulb were illuminated to recruit additional glomeruli. These results support a model of olfactory processing that is dependent upon mitral cell convergence and integration onto cortical cells. More broadly, these findings demonstrate a system for precise manipulation of neural activity in the intact mammalian brain with light and illustrate the use of ChR2 mice in exploring functional connectivity of complex neural circuits in vivo.
Epilepsy is a devastating disease, currently treated with medications, surgery or electrical stimulation. None of these approaches is totally effective and our ability to control seizures remains limited and complicated by frequent side effects. The emerging revolutionary technique of optogenetics enables manipulation of the activity of specific neuronal populations in vivo with exquisite spatiotemporal resolution using light. We used optogenetic approaches to test the role of hippocampal excitatory neurons in the lithium-pilocarpine model of acute elicited seizures in awake behaving rats. Hippocampal pyramidal neurons were transduced in vivo with a virus carrying an enhanced halorhodopsin (eNpHR), a yellow light activated chloride pump, and acute seizure progression was then monitored behaviorally and electrophysiologically in the presence and absence of illumination delivered via an optical fiber. Inhibition of those neurons with illumination prior to seizure onset significantly delayed electrographic and behavioral initiation of status epilepticus, and altered the dynamics of ictal activity development. These results reveal an essential role of hippocampal excitatory neurons in this model of ictogenesis and illustrate the power of optogenetic approaches for elucidation of seizure mechanisms. This early success in controlling seizures also suggests future therapeutic avenues.
The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. We used molecular genetic approaches to map the functional connectivity of a subpopulation of GABAergic neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-delta (PKCδ). Channelrhodopsin-2 assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKCδ+ neurons inhibit output neurons in the medial CE (CEm), and also make reciprocal inhibitory synapses with PKCδ− neurons in CEl. Electrical silencing of PKCδ+ neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus (CS), called CEloff units (Ciocchi et al, this issue). This correspondence, together with behavioral data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing.
Adult neurogenesis arises from neural stem cells within specialized niches1–3. Neuronal activity and experience, presumably acting upon this local niche, regulate multiple stages of adult neurogenesis, from neural progenitor proliferation to new neuron maturation, synaptic integration and survival1, 3. Whether local neuronal circuitry has a direct impact on adult neural stem cells is unknown. Here we show that in the adult hippocampus nestin-expressing radial glia-like quiescent neural stem cells4–9 (RGLs) respond tonically to the neurotransmitter GABA via γ2 subunit-containing GABAA Rs. Clonal analysis9 of individual RGLs revealed a rapid exit from quiescence and enhanced symmetric self-renewal after conditional γ2 deletion. RGLs are in close proximity to GAD67+ terminals of parvalbumin-expressing (PV+) interneurons and respond tonically to GABA released from these neurons. Functionally, optogenetic control of dentate PV+, but not somatostatin- or vasoactive intestinal polypeptide (VIP)-expressing, interneuron activity can dictate the RGL choice between quiescence and activation. Furthermore, PV+ interneuron activation restores RGL quiescence following social isolation, an experience that induces RGL activation and symmetric division8. Our study identifies a niche cell-signal-receptor trio and a local circuitry mechanism that control the activation and self-renewal mode of quiescent adult neural stem cells in response to neuronal activity and experience.
Inhibitory interneurons are essential components of the neural circuits underlying various brain functions. In the neocortex, a large diversity of GABAergic interneurons have been identified based on their morphology, molecular markers, biophysical properties, and innervation pattern1,2,3. However, how the activity of each subtype of interneurons contributes to sensory processing remains unclear. Here we show that optogenetic activation of parvalbumin-positive (PV+) interneurons in mouse V1 sharpens neuronal feature selectivity and improves perceptual discrimination. Using multichannel recording with silicon probes4,5 and channelrhodopsin 2 (ChR2)-mediated optical activation6, we found that elevated spiking of PV+ interneurons markedly sharpened orientation tuning and enhanced direction selectivity of nearby neurons. These effects were caused by the activation of inhibitory neurons rather than decreased spiking of excitatory neurons, since archaerhodopsin-3 (Arch)-mediated optical silencing7 of calcium/calmodulin-dependent protein kinase IIα-positive (CaMKIIα+) excitatory neurons caused no significant change in V1 stimulus selectivity. Moreover, the improved selectivity specifically required PV+ neuron activation, since activating somatostatin (SOM+) or vasointestinal peptide (VIP+) interneurons had no significant effect. Notably, PV+ neuron activation in awake mice caused a significant improvement in their orientation discrimination, mirroring the sharpened V1 orientation tuning. Together, these results provide the first demonstration that visual coding and perception can be improved by elevated spiking of a specific subtype of cortical inhibitory interneurons.
Neural circuits of the basal ganglia are critical for motor planning and action selection1-3. Two parallel basal ganglia pathways have been described4, which are proposed to exert opposing influences on motor function5-7. According to this classical model, activation of the direct pathway facilitates movement and activation of the indirect pathway inhibits movement. However, more recent anatomical and functional evidence has called into question the validity of this hypothesis8-10. Because this model has never been empirically tested, the specific function of these circuits in behaving animals remains unknown. Here, we directly activated basal ganglia circuitry in vivo, using optogenetic control11-14 of direct- and indirect-pathway medium spiny projection neurons (MSNs), achieved through Cre-dependent viral expression of channelrhodopsin-2 in the striatum of D1-Cre and D2-Cre BAC transgenic mice. Bilateral excitation of indirect-pathway MSNs elicited a parkinsonian state, distinguished by increased freezing, bradykinesia, and decreased locomotor initiations. In contrast, activation of direct-pathway MSNs reduced freezing and increased locomotion. In a mouse model of Parkinson's disease, direct pathway activation completely rescued deficits in freezing, bradykinesia, and locomotor initiation. Taken together, our findings establish a critical role for basal ganglia circuitry in the bidirectional regulation of motor behavior and indicate that modulation of direct pathway circuitry may represent an effective therapeutic strategy for ameliorating parkinsonian motor deficits.
Here we characterize several new lines of transgenic mice useful for optogenetic analysis of brain circuit function. These mice express optogenetic probes, such as enhanced halorhodopsin or several different versions of channelrhodopsins, behind various neuron-specific promoters. These mice permit photoinhibition or photostimulation both in vitro and in vivo. Our results also reveal the important influence of fluorescent tags on optogenetic probe expression and function in transgenic mice.
optogenetics; channelrhodopsin; photostimulation; photoinhibition; cerebellum; cortex; hippocampus; pons
A specific memory is thought to be encoded by a sparse population of neurons1,2. These neurons can be tagged during learning for subsequent identification3 and manipulation4,5,6. Moreover, their ablation or inactivation results in reduced memory expression, suggesting their necessity in mnemonic processes. However, a critical question of sufficiency remains: can one elicit the behavioral output of a specific memory by directly activating a population of neurons that was active during learning? Here we show that optogenetic reactivation of hippocampal neurons activated during fear conditioning is sufficient to induce freezing behavior. We labeled a population of hippocampal dentate gyrus neurons activated during fear learning with channelrhodopsin-2 (ChR2)7,8 and later optically reactivated these neurons in a different context. The mice showed increased freezing only upon light stimulation, indicating light-induced fear memory recall. This freezing was not detected in non-fear conditioned mice expressing ChR2 in a similar proportion of cells, nor in fear conditioned mice with cells labeled by EYFP instead of ChR2. Finally, activation of cells labeled in a context not associated with fear did not evoke freezing in mice that were previously fear conditioned in a different context, suggesting that light-induced fear memory recall is context-specific. Together, our findings indicate that activating a sparse but specific ensemble of hippocampal neurons that contribute to a memory engram is sufficient for the recall of that memory. Moreover, our experimental approach offers a general method of mapping cellular populations bearing memory engrams.
Dopamine D2 receptors (D2Rs) play a major role in the function of the prefrontal cortex (PFC), and may contribute to prefrontal dysfunction in conditions such as schizophrenia. Here we report that in mouse PFC, D2Rs are selectively expressed by a subtype of layer V pyramidal neurons that have thick apical tufts, prominent h-current, and subcortical projections. Within this subpopulation, the D2R agonist quinpirole elicits a novel afterdepolarization that generates voltage fluctuations and spiking for hundreds of milliseconds. Surprisingly, this afterdepolarization is masked in quiescent brain slices, but is readily unmasked by physiologic levels of synaptic input which activate NMDA receptors, possibly explaining why this phenomenon has not been reported previously. Notably, we could still elicit this afterdepolarization for some time after the cessation of synaptic stimulation. Besides NMDA receptors, the quinpirole-induced afterdepolarization also depended on L-type Ca2+ channels and was blocked by selective L-type antagonist nimodipine. To confirm that D2Rs can elicit this afterdepolarization by enhancing Ca2+ (and Ca2+-dependent) currents, we measured whole-cell Ca2+ potentials that occur after blocking Na+ and K+ channels, and found quinpirole enhanced these potentials, while the selective D2R antagonist (−)sulpiride had the opposite effect. Thus, D2Rs can elicit a Ca2+-channel dependent afterdepolarization that powerfully modulates activity in specific prefrontal neurons. Through this mechanism, D2Rs might enhance outputs to subcortical structures, contribute to reward related persistent firing, or increase the level of noise in prefrontal circuits.