High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice

To permit rapid optical control of brain activity, we have engineered multiple lines of transgenic mice that express the light-activated cation channel Channelrhodopsin-2 (ChR2) in subsets of neurons. Illumination of ChR2-positive neurons in brain slices produced photocurrents that generated action potentials within milliseconds and with precisely timed latencies. The number of light-evoked action potentials could be controlled by varying either the amplitude or duration of illumination. Furthermore, the frequency of light-evoked action potentials could be precisely controlled up to 30 Hz. Photostimulation also could evoke synaptic transmission between neurons, and, by scanning with a small laser light spot, we were able to map the spatial distribution of synaptic circuits connecting neurons within living cerebral cortex. We conclude that ChR2 is a genetically based photostimulation technology that permits analysis of neural circuits with high spatial and temporal resolution in transgenic mammals.     

An evaluation of causes for unreliability of synaptic transmission.

Transmission at individual synaptic contacts on CA1 hippocampal pyramidal neurons has been found to be very unreliable, with greater than half of the arriving presynaptic nerve impulses failing to evoke a postsynaptic response. This conclusion has been reached using the method of minimal stimulation of Schaffer collaterals and whole cell recording in hippocampal slices; with minimal stimulation only one or a few synapses are activated on the target neuron and the behavior of individual synapses can be examined. Four sources for the unreliability of synaptic transmission have been investigated: (i) the fluctuation of axon thresholds at the site of stimulation causing the failure to generate a nerve impulse in the appropriate Schaffer collaterals, (ii) the failure of nerve impulses generated at the site of stimulation to arrive at the synapse because of conduction failures at axon branch points, (iii) an artifactual synaptic unreliability due to performing experiments in vitro at temperatures well below the normal mammalian body temperature, and (iv) transmission failures due to probabilistic release mechanisms at synapses with a very low capacity to release transmitter. We eliminate the first three causes as significant contributions and conclude that probabilistic release mechanisms at low capacity synapses are the main cause of unreliability of synaptic transmission.     

Topographic specificity of functional connections from hippocampal CA3 to CA1

The hippocampus is a cortical region thought to play an important role in learning and memory. Most of our knowledge about the detailed organization of hippocampal circuitry responsible for these functions is derived from anatomical studies. These studies present an incomplete picture, however, because the functional character and importance of connections are often not revealed by anatomy. Here, we used a physiological method (photostimulation with caged glutamate) to probe the fine pattern of functional connectivity between the CA3 and CA1 subfields in the mouse hippocampal slice preparation. We recorded intracellularly from CA1 and CA3 pyramidal neurons while scanning with photostimulation across the entire CA3 subfield with high spatial resolution. Our results show that, at a given septotemporal level, nearby CA1 neurons receive synaptic inputs from neighboring CA3 neurons. Thus, the CA3 to CA1 mapping preserves neighbor relations.     

Synapse elimination accompanies functional plasticity in hippocampal neurons

A critical component of nervous system development is synapse elimination during early postnatal life, a process known to depend on neuronal activity. Changes in synaptic strength in the form of long-term potentiation (LTP) and long-term depression (LTD) correlate with dendritic spine enlargement or shrinkage, respectively, but whether LTD can lead to an actual separation of the synaptic structures when the spine shrinks or is lost remains unknown. Here, we addressed this issue by using concurrent imaging and electrophysiological recording of live synapses. Slices of rat hippocampus were cultured on multielectrode arrays, and the neurons were labeled with genes encoding red or green fluorescent proteins to visualize presynaptic and postsynaptic neuronal processes, respectively. LTD-inducing stimulation led to a reduction in the synaptic green and red colocalization, and, in many cases, it induced a complete separation of the presynaptic bouton from the dendritic spine. This type of synapse loss was associated with smaller initial spine size and greater synaptic depression but not spine shrinkage during LTD. All cases of synapse separation were observed without an accompanying loss of the spine during this period. We suggest that repeated low-frequency stimulation simultaneous with LTD induction is capable of restructuring synaptic contacts. Future work with this model will be able to provide critical insight into the molecular mechanisms of activity- and experience-dependent refinement of brain circuitry during development.     

Development of hippocampal mossy fiber synaptic outputs by new neurons in the adult brain

New neurons are continuously generated in restricted regions of the adult mammalian brain. Although these adult-born neurons have been shown to receive synaptic inputs, little is known about their synaptic outputs. Using retrovirus-mediated birth-dating and labeling in combination with serial section electron microscopic reconstruction, we report that mossy fiber en passant boutons of adult-born dentate granule cells form initial synaptic contacts with CA3 pyramidal cells within 2 weeks after their birth and reach morphologic maturity within 8 weeks in the adult hippocampus. Knockdown of Disrupted-in-Schizophrenia-1 (DISC1) in newborn granule cells leads to defects in axonal targeting and development of synaptic outputs in the adult brain. Together with previous reports of synaptic inputs, these results demonstrate that adult-born neurons are fully integrated into the existing neuronal circuitry. Our results also indicate a role for DISC1 in presynaptic development and may have implications for the etiology of schizophrenia and related mental disorders.     

Postsynaptic N-methyl-D-aspartate receptor function requires alpha-neurexins

Alpha-neurexins are neuron-specific cell-surface molecules that are essential for the functional organization of presynaptic Ca2+ channels and release sites. We have now examined postsynaptic glutamate receptor function in alpha-neurexin knockout (KO) mice by using whole-cell recordings in cultured neocortical slices. Unexpectedly, we find that alpha-neurexins are required for normal activity of N-methyl-D-aspartate (NMDA)- but not alpha-amino-3-hydroxy-5-methyl-4-isoxyzolepropionic acid (AMPA)-type glutamate receptors. In alpha-neurexin-deficient mice, the ratio of NMDA- to AMPA-receptor currents, recorded as evoked synaptic responses, was diminished approximately 50%. Furthermore, the NMDA-receptor-dependent component of spontaneous synaptic miniature responses was reduced approximately 50%, whereas the AMPA-receptor-dependent component was unaffected. No alterations in the levels of NMDA- or AMPA-receptor proteins were detected. These results suggest that alpha-neurexins are required to maintain normal postsynaptic NMDA-receptor function. The decrease in NMDA-receptor activity in alpha-neurexin-deficient synapses could be due to a transsynaptic effect on the postsynaptic neuron (i.e., alpha-neurexins on the presynaptic inputs guide postsynaptic NMDA-receptor function) or to a cell-autonomous postsynaptic effect of alpha-neurexins on NMDA-receptor activity. To distinguish between these two possibilities, we cocultured WT GFP-labeled neurons with neocortical slices from alpha-neurexin-deficient or control mice. No difference was found between WT neurons innervated by inputs that contained or lacked alpha-neurexins, indicating that the absence of presynaptic alpha-neurexins alone does not depress postsynaptic NMDA-receptor function. Our data suggest that, in addition to the previously described presynaptic impairments, loss of alpha-neurexins induces postsynaptic changes by a cell-autonomous mechanism.     

Rapid inhibition of neurons in the dorsal motor nucleus of the vagus by leptin

The peptide leptin conveys the availability of adipose energy stores to the brain. Increasing evidence implicates a significant role for extrahypothalamic sites of leptin action, including the dorsal vagal complex, a region critical for regulating visceral parasympathetic function. The hypothesis that leptin suppresses cellular activity in the dorsal motor nucleus of the vagus nerve (DMV) was tested using whole-cell patch-clamp recordings in brainstem slices. Leptin caused a rapid membrane hyperpolarization in 50% of rat DMV neurons. Leptin also hyperpolarized a subset of gastric-related neurons (62%), identified after gastric inoculation with a transneuronal retrograde viral tracer. The hyperpolarization was associated with a decrease in input resistance and cellular responsiveness and displayed characteristics consistent with an increased K+ conductance. Perfusion of tolbutamide (200 microM) reversed the leptin-induced hyperpolarization, and tolbutamide or wortmannin (10-100 nM) prevented the hyperpolarization, indicating that leptin activated an ATP-sensitive K+ channel via a phosphoinositide-3-kinase-dependent mechanism. Leptin reduced the frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs), whereas inhibitory postsynaptic currents (IPSCs) were largely unaffected. Electrical stimulation of the nucleus tractus solitarii (NTS) resulted in constant-latency EPSCs, which were decreased in amplitude by leptin. The paired-pulse ratio was increased, suggesting leptin effects involved activation of receptors presynaptic to the recorded neuron. A leptin-induced suppression of EPSCs, but not IPSCs, evoked by focal photolytic uncaging of glutamate within the NTS was also observed, supportive of leptin effects on the glutamatergic NTS projection to the DMV. Therefore, leptin directly hyperpolarized and indirectly suppressed excitatory synaptic activity to DMV neurons involved in visceral regulation, including gastric-related neurons.     

Very short-term plasticity in hippocampal synapses

Hippocampal pyramidal neurons often fire in bursts of action potentials with short interspike intervals (2–10 msec). These high-frequency bursts may play a critical role in the functional behavior of hippocampal neurons, but synaptic plasticity at such short times has not been carefully studied. To study synaptic modulation at very short time intervals, we applied pairs of stimuli with interpulse intervals ranging from 7 to 50 msec to CA1 synapses isolated by the method of minimal stimulation in hippocampal slices. We have identified three components of short-term paired-pulse modulation, including (i) a form of synaptic depression manifested after a prior exocytotic event, (ii) a form of synaptic depression that does not depend on a prior exocytotic event and that we postulate is based on inactivation of presynaptic N-type Ca²⁺ channels, and (iii) a dependence of paired-pulse facilitation on the exocytotic history of the synapse.     

Reduced gap junctional coupling leads to uncorrelated motor neuron firing and precocious neuromuscular synapse elimination

During late embryonic and early postnatal life, neuromuscular junctions undergo synapse elimination that is modulated by patterns of motor neuron activity. Here, we test the hypothesis that reduced spinal neuron gap junctional coupling decreases temporally correlated motor neuron activity that, in turn, modulates neuromuscular synapse elimination, by using mutant mice lacking connexin 40 (Cx40), a developmentally regulated gap junction protein expressed in motor and other spinal neurons. In Cx40-/- mice, electrical coupling among lumbar motor neurons, measured by whole-cell recordings, was reduced, and single motor unit recordings in awake, behaving neonates showed that temporally correlated motor neuron activity was also reduced. Immunostaining and intracellular recording showed that the neuromuscular synapse elimination was accelerated in muscles from Cx40-/- mice compared with WT littermates. Our work shows that gap junctional coupling modulates neuronal activity patterns that, in turn, mediate synaptic competition, a process that shapes synaptic circuitry in the developing brain.     

Dendro-dendritic bundling and shared synapses between gonadotropin-releasing hormone neurons

The pulsatile release of gonadotropin-releasing hormone (GnRH) is critical for mammalian fertility, but the mechanisms underlying the synchronization of GnRH neurons are unknown. In the present study, the full extent of the GnRH neuron dendritic tree was visualized by patching and filling individual GnRH neurons with biocytin in acute brain slices from adult GnRH-green fluorescent protein (GFP) transgenic mice. Confocal analysis of 42 filled GnRH neurons from male and female adult mice revealed that the dendrites of the great majority of GnRH neurons (86%) formed multiple close appositions with dendrites of other GnRH neurons. Two types of interactions were encountered; the predominant interaction was one of vertical dendritic bundling where dendrites were found to wrap around each other in the same axis. The other interaction was one in which a GnRH neuron dendrite intercepted other GnRH neuron dendrites in a perpendicular fashion. Electron microscopy using pre-embedded, silver-enhanced immunogold labeling for both GnRH and GFP peptides in GnRH-GFP transgenic mice, confirmed that GnRH neuron dendrites were often immediately juxtaposed. Membrane specializations, including punctae and zonula adherens, were found connecting adjacent dendritic elements of GnRH neurons. Remarkably, individual afferent axon terminals were found to synapse with multiple GnRH neuron dendrites at sites of bundling. Together, these data demonstrate that GnRH neurons are not isolated from one another but, rather, interconnected via their long dendritic extensions. The observation of shared synaptic input to bundled GnRH neuron dendrites suggests a mechanism of GnRH neuron synchronization.     

Three-dimensional imaging and photostimulation by remote-focusing and holographic light patterning

Access to three-dimensional structures in the brain is fundamental to probe signal processing at multiple levels, from integration of synaptic inputs to network activity mapping. Here, we present an optical method for independent three-dimensional photoactivation and imaging by combination of digital holography with remote-focusing. We experimentally demonstrate compensation of spherical aberration for out-of-focus imaging in a range of at least 300 μm, as well as scanless imaging along oblique planes. We apply this method to perform functional imaging along tilted dendrites of hippocampal pyramidal neurons in brain slices, after photostimulation by multiple spots glutamate uncaging. By bringing extended portions of tilted dendrites simultaneously in-focus, we monitor the spatial extent of dendritic calcium signals, showing a shift from a widespread to a spatially confined response upon blockage of voltage-gated Na(+) channels.     

ERK activation in axonal varicosities modulates presynaptic plasticity in the CA3 region of the hippocampus through synapsin I

Activity-dependent changes in the strength of synaptic connections in the hippocampus are central for cognitive processes such as learning and memory storage. In this study, we reveal an activity-dependent presynaptic mechanism that is related to the modulation of synaptic plasticity. In acute mouse hippocampal slices, high-frequency stimulation (HFS) of the mossy fiber (MF)-CA3 pathway induced a strong and transient activation of extracellular-regulated kinase (ERK) in MF giant presynaptic terminals. Remarkably, pharmacological blockade of ERK disclosed a negative role of this kinase in the regulation of a presynaptic form of plasticity at MF-CA3 contacts. This ERK-mediated inhibition of post-tetanic enhancement (PTE) of MF-CA3 synapses was both frequency- and pathway-specific and was observed only with HFS at 50 Hz. Importantly, blockade of ERK was virtually ineffective on PTE of MF-CA3 synapses in mice lacking synapsin I, 1 of the major presynaptic ERK substrates, and triple knockout mice lacking all synapsin isoforms displayed PTE kinetics resembling that of wild-type mice under ERK inhibition. These findings reveal a form of short-term synaptic plasticity that depends on ERK and is finely tuned by the firing frequency of presynaptic neurons. Our results also demonstrate that presynaptic activation of the ERK signaling pathway plays part in the activity-dependent modulation of synaptic vesicle mobilization and transmitter release.     

Distribution of calcium and potassium in presynaptic nerve terminals from cerebellar cortex

The elemental composition of the presynaptic nerve terminals in rapidly frozen synapses of the cerebellar molecular layer was determined by electron probe x-ray microanalysis and elemental imaging of characteristic x-rays. Elemental imaging of thin freeze-dried cryosections from fresh cerebellar slices frozen within 20 sec of removal from the brain showed normal concentrations of potassium (95 +/- 6 mmol/liter wet tissue +/- SEM) and calcium (0.8 +/- 0.4 mmol/liter) in whole presynaptic terminals, even though mitochondrial and nonmitochondrial sites containing up to 30 mmol of calcium per liter were present elsewhere in the neuropil. Quantitative electron probe analysis of synaptic vesicle clusters and intraterminal mitochondria indicated that their calcium concentrations were 0.4 +/- 0.1 and 1.2 +/- 0.2 mmol/liter, respectively. The low calcium content of presynaptic organelles was confirmed by the absence of detectable deposits in preparations freeze-substituted so as to stabilize calcium content. Similar experiments were carried out on cerebellar slices rapidly frozen after incubation in vitro. The distribution of potassium and calcium in presynaptic terminals of resting and depolarized (55 mM potassium) slices was qualitatively and quantitatively similar to that in freshly excised cortex, although resting slices lacked the few calcium-rich sites that appeared in other areas of the neuropil after stimulation. The calcium concentrations in whole terminals, synaptic vesicles, and mitochondria of resting slices were 1.4 +/- 0.7, 0.7 +/- 0.2, and 0.9 +/- 0.2 mmol/liter, respectively. Thus, amounts of calcium typical of storage organelles in other tissues are not present within cerebellar synaptic vesicles, suggesting that they have a limited role in calcium storage and release.     

A presynaptic locus for long-term potentiation of elementary synaptic transmission at mossy fiber synapses in culture.

The complex circuitry of the CA3 region and the abundance of collateral connections has made it difficult to study the mossy fiber pathway in hippocampal slices and therefore to establish the site of expression of long-term potentiation at these synapses. Using a novel cell culture system, we have produced long-term potentiation of the elementary synaptic connections on single CA3 pyramidal neurons following tetanic stimulation of individual dentate gyrus granule cells. As is the case for the hippocampal slice, this potentiation was independent of N-methyl-D-aspartate receptor activation, was simulated by application of forskolin, and its induction did not require any modulatory input. The increase in synaptic strength was accompanied by a reduction in the number of failures of transmission and by an increase in the coefficient of variation of the responses and was prevented by presynaptic injection of an inhibitor of protein kinase A. These findings show that mossy fiber long-term potentiation has a presynaptic locus and that its expression is dependent on protein kinase A.     

Readily releasable pool size changes associated with long term depression

We have estimated, for hippocampal neurons in culture, the size of the autaptic readily releasable pool before and after stimulation of the sort that produces culture long term depression (LTD). This stimulation protocol causes a decrease in the pool size that is proportional to the depression of synaptic currents. To determine if depression in this system is synapse specific rather than general, we have also monitored synaptic transmission between pairs of cultured hippocampal neurons that are autaptically and reciprocally interconnected. We find that the change in synaptic strength is restricted to the synapses on the target neuron that were active during LTD induction. When viewed from the perspective of the presynaptic neuron, however, synapse specificity is partial rather than complete: synapses active during induction that were not on the target neuron were partially depressed.     

Intracellular dye-marked enkephalin neurons in the magnocellular preoptic nucleus of the goldfish hypothalamus.

A method that combines intracellular recording, dye marking, and immunocytochemistry makes the study of functional and morphological aspects of enkephalin neurons in the magnocellular preoptic nucleus of the goldfish hypothalamus feasible. By use of multiple techniques, enkephalin neurons can be distinguished from other brain cells and can be reconstructed from drawings of serial sections containing the dye-injected opioid cells. These enkephalin cells and their processes measure 14-42 micron in somata diameter and are unipolar, bipolar, or multipolar. Their electrophysiological properties match those of other mammalian and fish magnocellular endocrine cells. This report confirms the one neuron-one hormone (peptide) hypothesis, supports synaptic over electronic coupling between enkephalin and adjacent hypothalamic neurons, and suggests that chemical and functional classification of single electrophysiologically and neuroanatomically studied central neurons can be achieved.     

Presynaptic modulation of voltage-dependent Ca2+ current: Mechanism for behavioral sensitization in Aplysia californica

Behavioral sensitization of the gill-withdrawal reflex of Aplysia is the result of a prolonged increase in transmitter release from the presynaptic terminals of sensory neurons. Earlier work suggested that this presynaptic facilitation might be mediated by a serotonin-sensitive adenylate cyclase in the sensory neuron terminals. Here we present evidence that presynaptic facilitation results from a cyclic AMP-dependent increase in the calcium current that underlies action potentials in the sensory neurons. The action potentials of sensory neuron cell bodies have, in addition to a sodium current, a calcium current that is enhanced by blocking the opposing potassium current with tetraethylammonium. Under these conditions, the action potentials show a slowly repolarizing plateau that follows the Nernst potential for a calcium electrode and serves as a sensitive assay for changes in calcium current. Stimulation of the pathway that mediates sensitization, incubation with serotonin or phosphodiesterase inhibitors, or intracellular injection of cyclic AMP produces an increase in the calcium plateau in the presence of tetraethylammonium. In addition, both before and after sensitizing stimulation, the duration of the plateau potential parallels transmitter release as measured by the amplitude of monosynaptic excitatory postsynaptic potentials evoked in the motor neurons by intracellular stimulation of single sensory neurons. These results are consistent with the idea that presynaptic facilitation is caused by a cyclic AMP-mediated increase in a voltage-sensitive calcium current in sensory neuron presynaptic terminals. This synaptic action is novel in that it can produce little or no change in the resting potential, is of long duration, and exerts its influence directly on a conductance triggered by the action potential, rather than on non-voltage-sensitive conductances, as is typical of conventional synaptic actions.     

Ethanol modulation of excitatory and inhibitory synaptic transmission in rat and monkey dentate granule neurons

BACKGROUND: The physiological mechanisms underlying the behavioral and cognitive effects of ethanol are not fully understood. However, there is now compelling evidence that ethanol acts, at least in part, by modulating the function of a small group of proteins that mediate excitatory and inhibitory synaptic transmission. For example, intoxicating concentrations of ethanol have been shown to enhance GABAergic synaptic inhibition and depress glutamatergic excitatory neurotransmission in a number of brain regions. Because all of these electrophysiological studies have been performed in rodent brain slice or neuronal culture preparations, direct evidence that ethanol exerts similar effects on synaptic transmission in the primate central nervous system is lacking. METHODS: We have therefore developed methods to perform patch-clamp electrophysiological recordings from neurons in acutely prepared monkey (Macaca fascicularis) hippocampal slices. We have used these methods to compare the acute effects of ethanol on excitatory and inhibitory synaptic transmission in rat and monkey dentate granule neurons. RESULTS: Under our recording conditions, ethanol significantly potentiated gamma-aminobutyric acid type A inhibitory postsynaptic currents in both rat and monkey neurons. In addition, ethanol significantly inhibited NMDA, but not AMPA, excitatory postsynaptic currents in dentate granule neurons from both species. Notably, no significant differences were observed in any of the pharmacological properties of inhibitory or excitatory synaptic responses recorded from rat and monkey neurons. CONCLUSIONS: These data suggest that the differences in the behavioral effects of ethanol that have been observed between rats and higher-order mammals, such as monkeys and humans, may not reflect differences in the sensitivity of some of the major synaptic sites of ethanol action. Moreover, our results provide empirical evidence for the use of rodent brain slice preparations in elucidating synaptic mechanisms of ethanol action in the primate central nervous system.     

Developmental hearing loss eliminates long-term potentiation in the auditory cortex

Severe hearing loss during early development is associated with deficits in speech and language acquisition. Although functional studies have shown a deafness-induced alteration of synaptic strength, it is not known whether long-term synaptic plasticity depends on auditory experience. In this study, sensorineural hearing loss (SNHL) was induced surgically in developing gerbils at postnatal day 10, and excitatory synaptic plasticity was examined subsequently in a brain slice preparation that preserves the thalamorecipient auditory cortex. Extracellular stimuli were applied at layer 6 (L6), whereas evoked excitatory synaptic potentials (EPSPs) were recorded from L5 neurons by using a whole-cell current clamp configuration. In control neurons, the conditioning stimulation of L6 significantly altered EPSP amplitude for at least 1 h. Approximately half of neurons displayed long-term potentiation (LTP), whereas the other half displayed long-term depression (LTD). In contrast, SNHL neurons displayed only LTD after the conditioning stimulation of L6. Finally, the vast majority of neurons recorded from control prehearing animals (postnatal days 9-11) displayed LTD after L6 stimulation. Thus, normal auditory experience may be essential for the maturation of synaptic plasticity mechanisms.