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Selectively activating (by optogenetics) parvalbumin-expressing (PV) interneurons induces GABA release onto CA1 pyramidal cells. Here we report that this release was attenuated by presynaptic mu opioid receptors (MORs) activation. On the other hand, conventional electric shock, presumably activating non-selectively presynaptic GABAergic terminals, also induced GABA release; however, this release showed relatively limited depression by MORs activation. The data suggest that MORs specifically inhibit GABA release from PV terminals and therefore, further support the idea that MORs contribute to homeostasis in CA1 neuro-circuit.

The spinal cord is engaged in all forms of motor performance but its functions are far from understood. Because network connectivity defines function, we explored the connectivity for muscular, tendon and tactile sensory inputs among a wide population of spinal interneurons in the lower cervical segments. Using low noise intracellular whole cell recordings in the decerebrated, nonanesthetized cat in vivo, we could define mono-, di-, trisynaptic inputs as well as the weights of each input. Whereas each neuron had a highly specific input, and each indirect input could moreover be explained by inputs in other recorded neurons, we unexpectedly also found the input connectivity of the spinal interneuron population to form a continuum. Our data hence contrasts with the currently widespread notion of distinct classes of interneurons. We argue that this suggested diversified physiological connectivity, which likely requires a major component of circuitry learning, implies a more flexible functionality.1.Graphical Abstract2.HighlightsIn vivo whole cell, intracellular recording of spinal interneurons.Patterns of input from Ia, Ib and cutaneous afferents is highly diversified.Learning appears to be a defining factor of spinal interneuron connectivity.

Summary Inhibitory interneurons govern virtually all computations in neocortical circuits and are in turn controlled by neuromodulation. While a detailed understanding of the distinct marker expression, physiology, and neuromodulator responses of different interneuron types exists for rodents and recent studies have highlighted the role of specific interneurons in converting rapid neuromodulatory signals into altered sensory processing during locomotion, attention, and associative learning, it remains little understood whether similar mechanisms exist in human neocortex. Here, we use whole-cell recordings combined with agonist application, transgenic mouse lines, in situ hybridization, and unbiased clustering to directly determine these features in human layer 1 interneurons (L1-INs). Our results indicate pronounced nicotinic recruitment of all L1-INs, whereas only a small subset co-expresses the ionotropic HTR3 receptor. In addition to human specializations, we observe two comparable physiologically and genetically distinct L1-IN types in both species, together indicating conserved rapid neuromodulation of human neocortical circuits through layer 1., Graphical Abstract, Highlights • Layer 1 interneurons in human and mouse neocortex respond strongly to acetylcholine • These rapid responses are mediated by α7 and β2-containing nicotinic receptors • Human layer 1 comprises neurogliaform cells expressing the conserved marker Ndnf • Apart from conserved features, human L1 interneurons show a number of specializations, Inhibitory interneurons govern the function of neural circuits and are in turn controlled by neuromodulation. Here, Poorthuis et al. demonstrate that these mechanisms are conserved in layer 1 of human neocortex, where interneurons express nicotinic acetylcholine receptors that mediate fast responses and thereby enable reconfiguration of circuit function at rapid timescales.

Background Mutations in TSC1 or TSC2 genes cause tuberous sclerosis complex (TSC), a disorder associated with epilepsy, autism, and intellectual disability. TSC1 and TSC2 are repressors of the mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of protein synthesis. Dysregulation of mTORC1 in TSC mouse models leads to impairments in excitation-inhibition balance, synaptic plasticity, and hippocampus-dependent learning and memory deficits. However, synaptic inhibition arises from multiple types of inhibitory interneurons and how changes in specific interneurons contribute to TSC remains largely unknown. In the present work, we determined the effect of conditional Tsc1 haploinsufficiency in a specific subgroup of inhibitory cells on hippocampal function in mice. Methods We investigated the consequences of conditional heterozygous knockout of Tsc1 in MGE-derived inhibitory cells by crossing Nkx2.1Cre/wt;Tsc1f/f mice. We examined the changes in mTORC1 activity and synaptic transmission in hippocampal cells, as well as hippocampus-related cognitive tasks. Results We detected selective increases in phosphorylation of ribosomal protein S6 in interneurons, indicating cell-specific-upregulated mTORC1 signaling. At the behavioral level, Nkx2.1Cre/wt;Tsc1f/wt mice exhibited intact contextual fear memory, but impaired contextual fear discrimination. They displayed intact spatial learning and reference memory but impairment in spatial working memory. Whole-cell recordings in hippocampal slices of Nkx2.1Cre/wt;Tsc1f/wt mice showed intact basic membrane properties, as well as miniature excitatory and inhibitory synaptic transmission, in pyramidal and Nkx2.1-expressing inhibitory cells. Using optogenetic activation of Nkx2.1 interneurons in slices of Nkx2.1Cre/wt;Tsc1f/wt mice, we found a decrease in synaptic inhibition of pyramidal cells. Chronic, but not acute treatment, with the mTORC1 inhibitor rapamycin reversed the impairment in synaptic inhibition. Conclusions Our results indicate that Tsc1 haploinsufficiency in MGE-derived inhibitory cells upregulates mTORC1 activity in these interneurons, reduces their synaptic inhibition of pyramidal cells, and alters contextual fear discrimination and spatial working memory. Thus, selective dysregulation of mTORC1 function in Nkx2.1-expressing inhibitory cells appears sufficient to impair synaptic inhibition and contributes to cognitive deficits in the Tsc1 mouse model of TSC.

Summary The lateral hypothalamic area (LH) is a vital controller of arousal, feeding, and metabolism [1, 2], which integrates external and internal sensory information. Whereas sensory and whole-body output properties of LH cell populations have received much interest, their intrinsic synaptic organization has remained largely unstudied. Local inhibitory and excitatory connections could help integrate and filter sensory information and mutually inhibitory connections [3] could allow coordinating activity between LH cell types, some of which have mutually exclusive behavioral effects, such as LH VGLUT2 and VGAT neurons [4, 5, 6, 7] and orexin- (ORX) and melanin-concentrating hormone (MCH) neurons [8, 9, 10]. However, classical Golgi staining studies did not find interneurons with locally ramifying axons in the LH [11, 12], and nearby subthalamic and thalamic areas lack local synaptic connectivity [13, 14]. Studies with optogenetic circuit mapping within the LH have demonstrated only a minority of connections when a large pool of presynaptic neurons was activated [15, 16, 17, 18, 19]. Because multiple patch clamp has not been used to study LH connectivity, aside from a limited dataset of MCH neurons where no connections were discovered [15], we used quadruple whole-cell recordings to screen connectivity within the LH with standard methodology we previously used in the neocortex [20, 21, 22]. Finding a lack of local connectivity, we used optogenetic circuit mapping to study the strength of LH optogenetic responses and network oscillations, which were consistent with ultra-sparse intrinsic connectivity within the LH. These results suggest that input from other brain structures is decisive for selecting active populations in the LH., Graphical Abstract, Highlights • LH neurons within, It is unknown how neural activity is coordinated among lateral hypothalamic area (LH) neurons, which generate fundamental behavioral actions such as attack and evasion. Burdakov and Karnani show there is near-zero local connectivity in the LH, suggesting that incoming synaptic input is integrated primarily within individual neurons.

Graphical abstract, Understanding how neuromodulators influence synaptic transmission and intrinsic excitability within the entorhinal cortex (EC) is critical to furthering our understanding of the molecular and cellular aspects of this region. Organotypic cultures can provide a cost-effective means to employ selective molecular biological strategies in elucidating cellular mechanisms of neuromodulation in the EC. We therefore adapted our acute slice model for organotypic culture applications and optimized a protocol for the preparation and biolistic transfection of cultured horizontal EC slices. Here, we present our detailed protocol for culturing EC slices. Using an n-methyl-d-glucamine (NMDG)-containing cutting solution, we obtain healthy EC slice cultures for electrophysiological recordings. We also present our protocol for the preparation of “bullets” carrying one or more constructs and demonstrate successful transfection of EC slices. We build upon previous methods and highlight specific aspects in our method that greatly improved the quality of our results. We validate our methods using immunohistochemical, imaging, and electrophysiological techniques. The novelty of this method is that it provides a description of culturing and transfection of EC neurons for specifically addressing their functionality. This method will enable researchers interested in entorhinal function to quickly adopt a similar slice culture transfection system for their own investigations.

Cortical neurons process information on a background of spontaneous, ongoing activity with distinct spatiotemporal profiles defining different cortical states. During wakefulness, cortical states alter constantly in relation to behavioral context, attentional level or general motor activity. In this review article, we will discuss our current understanding of cortical states in awake rodents, how they are controlled, their impact on sensory processing, and highlight areas for future research. A common observation in awake rodents is the rapid change in spontaneous cortical activity from high-amplitude, low-frequency (LF) fluctuations, when animals are quiet, to faster and smaller fluctuations when animals are active. This transition is typically thought of as a change in global brain state but recent work has shown variation in cortical states across regions, indicating the presence of a fine spatial scale control system. In sensory areas, the cortical state change is mediated by at least two convergent inputs, one from the thalamus and the other from cholinergic inputs in the basal forebrain. Cortical states have a major impact on the balance of activity between specific subtypes of neurons, on the synchronization between nearby neurons, as well as the functional coupling between distant cortical areas. This reorganization of the activity of cortical networks strongly affects sensory processing. Thus cortical states provide a dynamic control system for the moment-by-moment regulation of cortical processing.

Cerebellar granule cells (GrCs) constitute over half of all neurons in the vertebrate brain and are proposed to decorrelate convergent mossy fiber inputs in service of learning. Interneurons within the granule cell layer, Golgi cells (GoCs), are the primary inhibitors of this vast population and therefore play a major role in influencing the computations performed within the layer. Despite this central function for GoCs, few studies have directly examined how GoCs integrate inputs from specific afferents which vary in density to regulate GrC population activity. We used a variety of methods in mice of either sex to study feedforward inhibition recruited by identified MFs, focusing on features that would influence integration by GrCs. Comprehensive 3D reconstruction and quantification of GoC axonal boutons revealed tightly clustered boutons that focus feedforward inhibition in the neighborhood of GoC somata. Acute whole cell patch clamp recordings from GrCs in brain slices showed that despite high bouton density, fast phasic inhibition was very sparse relative to slow spillover mediated inhibition. Furthermore, dynamic clamp simulating inhibition combined with optogenetic mossy fiber activation supported the predominant role of slow spillover mediated inhibition. Whole cell recordings from GoCs revealed a role for the density of active MFs in preferentially driving them. Thus, our data provide empirical conformation of predicted rules by which MFs activate GoCs to regulate GrC activity levels.

Summary GABAB receptor (GABABR) autoantibodies have been detected in the serum of immunotherapy‐responsive patients with autoimmune encephalitis. This study aimed to investigate the effect of immunoglobulin G (IgG) from a patient with GABABR antibodies on primary neuronal cultures and acute slices of entorhinal cortex. Primary hippocampal neuronal cultures were incubated with serum immunoglobulin from patients with GABABR or AMPA receptor (AMPAR) antibodies for up to 72 h to investigate their effect on receptor surface expression. Whole‐cell patch‐clamp recordings from layer III pyramidal cells of the medial entorhinal cortex were used to examine the effect on neuronal activity. GABABR surface expression was unaltered by incubation with GABABR antibodies. By contrast, after 24 h application of AMPAR antibodies, AMPARs were undetectable. However, acute application of GABABR IgG decreased both the duration of network UP states and the spike rate of pyramidal cells in the entorhinal cortex. GABABR antibodies do not appear to affect GABABRs by internalization but rather reduce excitability on the medial temporal lobe networks. This unusual mechanism of action may be exploited in rational drug development strategies.

Timing is a crucial aspect of synaptic integration. For pyramidal neurons that integrate thousands of synaptic inputs spread across hundreds of microns, it is thus a challenge to maintain the timing of incoming inputs at the axo-somatic integration site. Here we show that pyramidal neurons in the rodent hippocampus use a gradient of inductance in the form of hyperpolarization-activated cation-nonselective (HCN) channels as an active mechanism to counteract location-dependent temporal differences of dendritic inputs at the soma. Using simultaneous multi-site whole-cell recordings complemented by computational modeling, we find that this intrinsic biophysical mechanism produces temporal synchrony of rhythmic inputs in the theta and gamma frequency ranges across wide regions of the dendritic tree. While gamma and theta oscillations are known to synchronize activity across space in neuronal networks, our results identify a new mechanism by which this synchrony extends to activity within single pyramidal neurons with complex dendritic arbors.

This protocol provides a method for quadruple whole-cell recording to study synaptic plasticity of neocortical connections, with a special focus on spike-timing-dependent plasticity (STDP). It also describes how to morphologically identify recorded cells from two-photon laser-scanning microscopy (2PLSM) stacks.

The review deals largely with studies from my laboratory that were prompted by conversations I had with Gerhard Neuweiler more than 15 years ago. The studies were conducted on bats and dealt with mechanisms that enable the population of neurons in the inferior colliculus (IC) to respond selectively to the variety of signals bats emit for both communication and echolocation. The first section is concerned with how neurons in the dorsal nucleus of the lateral lemniscus (DNLL), the nucleus ventral to the IC, respond to species-specific signals and how they compare to responses of IC neurons evoked by the same signals. Those studies showed that DNLL neurons have no sideband inhibition and their responses are determined by excitation. In contrast, inhibition dominates in the IC where it carves out highly selective discharge properties. Those studies, in turn, raised questions about the quantitative features of inhibition that could only be answered with more sophisticated techniques. In the second section, results from analyses with spectrotemporal receptive fields (STRFs) are presented, and in the final section I show data derived from in vivo whole cell recordings that illustrate how features of inhibition interact with excitation to generate directionality selective responses in the IC.

Electrophysiology, the 'gold standard' for investigating neuronal signalling, is being challenged by a new generation of optical probes. Together with new forms of microscopy, these probes allow us to measure and control neuronal signals with spatial resolution and genetic specificity that already greatly surpass those of electrophysiology. We predict that the photon will progressively replace the electron for probing neuronal function, particularly for targeted stimulation and silencing of neuronal populations. Although electrophysiological characterization of channels, cells and neural circuits will remain necessary, new combinations of electrophysiology and imaging should lead to transformational discoveries in neuroscience.

Layer V pyramidal neurons are anatomically and physiologically heterogeneous and project to multiple intracortical and subcortical targets. However, because most physiological studies of layer V pyramidal neurons have been carried out on unidentified cells, we know little about how anatomical and physiological properties relate to subcortical projection site. Here we combine neuroanatomical tract tracing with whole cell recordings in mouse somatosensory cortex to test whether neurons with the same projection target form discrete subpopulations and whether they have stereotyped physiological properties. Our findings indicate that corticothalamic and -trigeminal neurons are two largely nonoverlapping subpopulations, whereas callosal and corticostriatal neurons overlap extensively. The morphology as well as the intrinsic membrane and firing properties of corticothalamic and corticotrigeminal neurons differ from those of callosal and corticostriatal neurons. In addition, we find that each class of projection neuron exhibits a unique compliment of hyperpolarizing and depolarizing afterpotentials that further suggests that cortical neurons with different subcortical targets are distinct from one another.

Tuning curves were recorded with patch electrodes from the inferior colliculus (IC) of awake bats to evaluate the tuning of the inputs to IC neurons, reflected in their synaptic tuning, compared with the tuning of their outputs, expressed in their discharge tuning. A number of unexpected features were revealed with whole-cell recordings. Among these was that most neurons responded to tones with inhibition and/or subthreshold excitation over a surprisingly broad frequency range. The synaptic tuning in many cells was at least 1.5–2.0 octaves wide and, on average, was more than twice as wide as the frequency range that evoked discharges even after inhibition was blocked. In most cells, tones evoked complex synaptic response configurations that varied with frequency, suggesting that these cells were not innervated by congruent excitatory and inhibitory projections. Synaptic tuning was not only wide but was also diverse, in which some cells were dominated by excitation (n= 20), others were dominated by excitation with sideband inhibition (n= 21), but most were dominated by inhibition with little evidence of excitation (n= 31). Another unexpected finding was that some cells responded with inhibition to the onset and offset of tones over a wide frequency range, in which the patterns of synaptic responses changed markedly with frequency. These cells never fired to tones at 50 dB sound pressure level but fired to frequency-modulated sweeps at that intensity and were directionally selective. Thus, the features revealed by whole-cell recordings show that the processing in many IC cells results from inputs spectrally broader and more complex than previously believed.

Locally projecting inhibitory interneurons play a crucial role in the patterning and timing of network activity. However, because of their relative inaccessibility, little is known about their development or incorporation into circuits. In this report we demonstrate that the GABAergic R-interneuron circuit undergoes a reorganization in the chick embryo spinal cord between embryonic days 8 and 15 (E8 and E15). R-interneurons receive synaptic input from and project back to motoneurons. By stimulating motoneurons projecting in one ventral root and recording the disynaptic response from motoneurons in adjacent segments, we show that the output of the R-interneuron circuit is reorganized during development. After stimulation of the LS2 ventral root, disynaptic responses observed in whole cell recordings became more common and stronger for LS3 motoneurons and less common for the more distant LS4 motoneurons from E8 to E10. Optical studies demonstrated that R-interneurons activated by LS2 stimulation were restricted to the LS2 segment and had a small glutamatergic component at both E8 and E10, but that more R-interneurons were activated within the segment by E10. The recruitment of more LS2 R-interneurons at E10 is likely to contribute to stronger projections to LS3 motoneurons, but the fact that fewer LS4 motoneurons receive this input is more consistent with a functional refinement of the more distant projection of the GABAergic R-interneuron. Interestingly, this pattern of reorganization was not observed throughout the rostrocaudal extent of the cord, introducing the possibility that refinement could serve to remove connections between functionally unrelated interneurons and motoneurons.

Current dogma holds that a canonical cortical circuit is formed by cellular elements that are basically identical across species. However, detailed and direct comparisons between species of specific elements of this circuit are limited in number. In this study, we compared the morphological and physiological properties of neurogliaform (NGF) inhibitory neurons in the prefrontal cortex (PFC) of macaque monkeys and rats. In both species, NGF cells were readily identified based on their distinctive morphological features. Indeed, monkey NGF cells had only a few morphological features that differed from rat, including a larger soma, a greater number of dendrites, and a more compact axonal field. In contrast, whole cell recordings of the responses to injected current steps revealed important differences between monkey and rat NGF cells. Monkey NGF cells consistently generated a short-latency first spike riding on an initial depolarizing hump, whereas in rat NGF cells, the first spike appeared after a substantial delay riding on a depolarizing ramp not seen in monkey NGF cells. Thus although rat NGF cells are traditionally classified as late-spiking cells, monkey NGF cells did not meet this physiological criterion. In addition, NGF cells in monkey appeared to be more excitable than those in rat because they displayed a higher input resistance, a lower spike threshold, and a higher firing frequency. Finally, NGF cells in monkey showed a more prominent spike-frequency adaptation as compared with rat. Our findings indicate that the canonical cortical circuit differs in at least some aspects of its constituent elements across species.

While electrophysiological recordings from visually identified cell bodies or dendrites are routinely performed in cell culture and acute brain slice preparations, targeted recordings from the mammalian nervous system are currently not possible in vivo. The “blind” approach that is used instead is somewhat random and largely limited to common neuronal cell types. This approach prohibits recordings from, for example, molecularly defined and/or disrupted populations of neurons. Here we describe a method, which we call TPTP (two-photon targeted patching), that uses two-photon imaging to guide in vivo whole-cell recordings to individual, genetically labeled cortical neurons. We apply this technique to obtain recordings from genetically manipulated, parvalbumin-EGFP-positive interneurons in the somatosensory cortex. We find that both spontaneous and sensory-evoked activity patterns involve the synchronized discharge of electrically coupled interneurons. TPTP applied in vivo will therefore provide new insights into the molecular control of neuronal function at the systems level.

A blind patch-clamp technique for in vivo whole-cell recordings in the intact brain is described. Recordings were obtained from various neuronal cell types located 100-5,000 microm from the cortical surface. Access resistance of recordings was as low as 10 M Omega but increased with recording depth and animal age. Recordings were remarkably stable and it was therefore possible to obtain whole-cell recordings in awake, head-fixed animals. The whole-cell configuration permitted rapid dialysis of cells with a calcium buffer. In most neurons very little ongoing action potential (AP) activity was observed and the spontaneous firing rates were up to 50-fold less than what has been reported by extracellular unit recordings. AP firing in the brain might therefore be far sparser than previously thought.

Synaptic transmission in pair recordings from CA3 pyramidal cells in organotypic culture. We performed simultaneous whole cell recordings from pairs of monosynaptically coupled hippocampal CA3 pyramidal neurons in organotypic slices. Stimulation of an action potential in a presynaptic cell resulted in an AMPA-receptor-mediated excitatory postsynaptic current (EPSC) in the postsynaptic cell that averaged ∼34 pA. The average size of EPSCs varied in amplitude over a 20-fold range across different pairs. Both paired-pulse facilitation and depression were observed in the synaptic current in response to two presynaptic action potentials delivered 50 ms apart, but the average usually was dominated by depression. In addition, the amplitude of the second EPSC depended on the amplitude of the first EPSC, indicating competition between successive events for a common resource that is not restored within the 50-ms interpulse interval. Variation in the synaptic strength among pairs could arise from a variety of sources. Our data from anatomic reconstruction, 1/CV2 analysis, paired-pulse analysis, and manipulations of calcium/magnesium ratio suggest that differences in quantal size and release probability do not appear to vary sufficiently to fully account for the observed differences in amplitude. Thus it seems most likely that the variability in EPSC amplitude between pairs arises primarily from differences in the number of functional synapses. Injections of the calcium chelator bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid into the presynaptic neuron resulted in a rapid and nearly complete block of transmission, whereas injection of the slower-acting chelator EGTA resulted in a variable and partial block. In addition to demonstrating the feasibility of manipulating the intracellular presynaptic environment by injection into the presynaptic soma, these data, and the EGTA results in particular may suggest variability in the linkage between calcium entry sites an release sites in these synapses.

Gigaseals were obtained from enzymatically dissociated epithelial cells of Hydra vulgaris, and resting potentials were recorded in the whole‐cell configuration. These results were obtained in spite of hard difficulties of applying patch‐clamp techniques in the whole‐cell configuration to Hydra cells. These difficulties are principally inherent to the fact that Hydra lives in a low ionic strength medium. It will be now possible to apply patch‐clamp techniques to the study of Hydra extraocular photoreception.

1. Whole cell recordings from neurons in the rostral, gustatory nucleus of the solitary tract (rNST) were made using the “blind” patch-clamp technique in horizontal brain stem slices of rats. 2. Postsynaptic potentials (PSP) were elicited in 71 rNST neurons by electrical stimulation of the solitary tract (ST). To investigate PSPs evoked by convergent input from the chorda tympani and glossopharyngeal nerves, the ST was stimulated at levels where these two nerves terminate. These are referred to as rostral (rST) and intermediate (iST) ST, respectively. 3. When the rST was stimulated 72% of the PSPs were depolarizing, and 28% were hyperpolarizing (n = 64). Stimulation of the intermediate ST resulted in 75% depolarizing and 25% hyperpolarizing PSPs (n = 56). 4. Application of gamma-aminobuturic acid-A (GABAA) and glutamate receptor blockers revealed that all PSPs recorded in the present study were a composite of summed excitatory and inhibitory PSPs. Application of the GABAA receptor blocker bicuculline, by eliminating the hyperpolarizing component of a PSP, revealed the excitatory postsynaptic potential (EPSP) component of the potential. Bicuculline also increased the amplitude and prolonged the decay time of the depolarizing potentials once the hyperpolarizing potential component had been eliminated. These pure EPSP revealed by GABAA receptor blockade reversed at approximately 0 mV. 5. Application of glutamate ionotropic receptor blockers effectively eliminated the initiation of the synaptic responses evoked by ST stimulation. If the stimulus strength was increased, an inhibitory postsynaptic potential (IPSP) was elicited, presumably by direct activation of interneurons close to the stimulating electrode. These IPSPs had a mean reversal potential of -88 mV. 6. When synaptic responses were initiated by stimulation of the projection areas of both the chorda tympani and glossopharyngeal nerves, all neurons tested (n = 49) responded to stimulation of both sites on the ST. The resulting synaptic potential was a sum of the two individual synaptic potentials. 7. If stimulation of the rostral and intermediate sites both elicited depolarizing potentials, the potential resulting from stimulation of both sites was the arithmetical sum of the two individual PSPs. The EPSPs summed even if the time between stimulation of the rostral and intermediate sites was separated by < or = 100 ms. 8. Inhibitory PSPs evoked by simultaneous stimulation of the rostral and intermediate ST also summed. The summation was not linear and saturated at a mean level of -66 mV. 9. When the PSP at one stimulation site was excitatory but inhibitory at the other site, the PSP wave form resulting from dual stimulation was a complex mixture of the two individual potentials. The inhibitory potential was capable of blocking action potentials resulting from the excitatory PSP. 10. These results indicate that synaptic responses in rNST are complex mixtures of excitatory and inhibitory potentials. The synaptic potentials result from excitatory afferent input mediated by glutamate and inhibitory input derived from interneurons. Inhibitory transmission is mediated primarily at GABAA synapses. Stimulation of the rNST afferent input derived from the chorda tympani and glossopharyngeal nerves reveals complex convergent input. The complexity of these synaptic interactions indicates that considerable processing of gustatory information occurs at the first central synapse in the taste pathway.

Patch-type pipettes increasingly are being used to obtain intracellular ‘whole-cell’ recording from neurons. Here we describe our methods for making whole-cell recordings in vivo from midbrain neurons in an electric fish. Novel elements in the procedure are: A device for micropositioning the pipette when near a cell use of a ‘Picospritzer’ for cleaning the pipette tip and cell surface, and an electroporetic method, for perforating the patch following seal formation. In addition, we show that extracellular and intracellular recordings can be made from the same neuron. Stable intracellular recordings can be made from neurons at least as small as 10 μm.

The in vitro whole spinal cord preparation has been an invaluable tool for the study of the neural network that underlies walking because it provides a means of recording fictive locomotor activity following surgical and/or pharmacological manipulation. The recent use of molecular genetic techniques to identify discrete neuronal populations in the spinal cord and subsequent studies showing some of these populations to be involved in locomotor activity have been exciting developments that may lead to a better understanding of the structure and mechanism of function of this neural network. It would be of great benefit if the in vitro whole spinal cord preparation could be updated to allow for the direct targeting of genetically defined neuronal populations, allowing each to be characterized physiologically and anatomically. This report describes a new technique that enables the visualization of, and targeted whole cell patch-clamp recordings from, genetically defined populations of neurons while leaving connectivity largely intact. The key feature of this technique is a small notch cut in the lumbar spinal cord that reveals cells located in the intermediate laminae while leaving the ventral portion of the spinal cord—the region containing the locomotor neural network—untouched. Whole cell patch-clamp recordings demonstrate that these neurons are healthy and display large rhythmic depolarizations that are related to electroneurogram bursts recorded from ventral roots during fictive locomotion. Intracellular labeling demonstrates that this technique can also be used to map axonal projection patterns of neurons. We expect that this procedure will greatly facilitate electrophysiological and anatomical study of important neuronal populations that constitute neural networks throughout the CNS.

Intracellular recordings are routinely used to study the synaptic and intrinsic properties of neurons in vitro. A key requirement for these recordings is a mechanically very stable preparation; thus their use in vivo had been limited previously to head-restrained animals. We have recently demonstrated that anchoring the electrode rigidly in place with respect to the skull provides sufficient stabilization for long-lasting, high-quality whole-cell recordings in awake, freely moving rats. This protocol describes our procedure in detail, adds specific instructions for targeting hippocampal CA1 pyramidal neurons and updates it with changes that facilitate patching and improve the success rate. The changes involve combining a standard, nonhead-mounted micromanipulator with a gripper to firmly hold the recording pipette during the anchoring process then gently release it afterwards. The procedure from the beginning of surgery to the end of a recording takes approximately 5 h. This technique allows new studies of the mechanisms underlying neuronal integration and cellular/synaptic plasticity in identified cells during natural behaviors.

Throughout the CNS, small conductance Ca(2+)-activated potassium (SK) channels modulate firing frequency and neuronal excitability. We have identified a novel, shorter isoform of standard SK2 (SK2-std) in mouse brain which we named SK2-sh. SK2-sh is alternatively spliced at exon 3 and therefore lacks 140 amino acids, which include transmembrane domains S3, S4 and S5, compared with SK2-std. Western blot analysis of mouse hippocampal tissue revealed a 47 kDa protein product as predicted for SK2-sh along with a 64 kDa band representing the standard SK2 isoform. Electrophysiological recordings from transiently expressed SK2-sh revealed no functional channel activity or interaction with SK2-std. With the help of real-time PCR, we found significantly higher expression levels of SK2-sh mRNA in cortical tissue from AD cases when compared with age-matched controls. A similar increase in SK2-sh expression was induced in cortical neurons from mice by cytokine exposure. Substantial clinical evidence suggests that excess cytokines are centrally involved in the pathogenesis of Alzheimer's disease. Thus, SK2-sh as a downstream target of cytokines, provide a promising target for additional investigation regarding potential therapeutic intervention.

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents. The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown. The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

Using whole cell recordings from antennal-lobe (AL) neurons in vitro and in situ, in semi-intact brain preparations, we examined membrane properties that contribute to electrical activity exhibited by developing neurons in primary olfactory centers of the brain of the sphinx moth, Manduca sexta. This activity is characterized by prolonged periods of membrane depolarization that resemble plateau potentials. The presence of plateau potential–generating mechanisms was confirmed using a series of tests established earlier. Brief depolarizing current pulses could be used to trigger a plateau state. Once triggered, plateau potentials could be terminated by brief pulses of hyperpolarizing current. Both triggering and terminating of firing states were threshold phenomena, and both conditions resulted in all-or-none responses. Rebound excitation from prolonged hyperpolarizing pulses could also be used to generate plateau potentials in some cells. These neurons were found to express a hyperpolarization-activated inward current. Neither the generation nor the maintenance of plateau potentials was affected by removal of Na+ions from the extracellular medium or by blockade of Na+currents with TTX. However, blocking of Ca2+currents with Cd2+(5 × 10−4M) inhibited the generation of plateau potentials, indicating that, in Manduca AL neurons, plateau potentials depend on Ca2+. Examining Ca2+currents in isolation revealed that activation of these currents occurs in the absence of experimentally applied depolarizing stimuli. Our results suggest that this activity underlies the generation of plateau potentials and characteristic bursts of electrical activity in developing AL neurons of M. sexta.

The complexity of the vertebrate brain poses an enormous challenge to experimental neuroscience. One way of dealing with this complexity has been to investigate different aspects of brain function in widely different preparations, each best suited to address a particular question. Accordingly

For synaptic and cellular analyses of the mammalian respiratory network, intracellular recording and good pharmacological access to respiratory neurones is required. Using an existing arterially perfused in situ preparation of neonatal rat, we report on a method allowing stable intracellular recordings of ventrolateral medullary respiratory neurones. The in situ preparation generates a recognizable eupnoeic respiratory motor pattern similar to that reported in vivo. Using this preparation, we have developed a methodology for the use of patch pipettes to record from neurones within the ventral respiratory group. This technique in the arterially perfused neonatal rat is novel and has the advantage that neurones can be recorded from an intact and well-oxygenated brainstem in which the pontine regions are known to be viable. We describe the methods, present the first whole cell recordings of numerous types of respiratory neurones from neonatal rats in such a preparation, and demonstrate the applicability of the model for neuropharmacological experiments.

Using whole cell recordings, I analyzed the intrinsic discharge properties for 285 neurons in Rexed's laminae III–V of isolated hamster spinal cord preparations. Neurons were characterized by their responses to step-wise and ramp-hold depolarizing current applied through the recording pipettes. Tonic cells (133/285; 47%) fired repetitively during step-wise current application. Firing decayed linearly (–0.14 to –4.3 imp · s–1 · s–1) or was bimodal, with an initial exponential phase (τ ≈ 450 ms) followed by a linear decline (–0.02 to –6.3 imp · s–1 · s–1); discharge frequency was unrelated to current trajectory. Phasic-firing cells (108/285; 38%) responded with a burst discharge having an initial rapid, exponential decrease (τ ≈ 30 ms) and subsequent linear decline (–1 to –78 imp · s–1 · s–1). Phasic cells were activated preferentially by fast current ramps (slope, 70 pA/s–2.2 nA/s) with the number and frequency of impulses increasing with current slope. Delayed-firing cells (44/285; 15%), responded to current steps with an accelerating firing following a substantial latent period (0.5–4 s) and discharged during current ramps with slopes less than ≈100 pA/s. Intracellular staining revealed a significant association between electrophysiological profile and neuronal morphology. A majority of presumed projection cells (22/30; 73%) exhibited tonic firing to step-wise activation. The preponderance of phasic and delayed firing cells, 93% (42/45) and 71% (12/17), respectively, were interneurons with local or intersegmental terminations. Differential sensitivity to static and time-varying components of membrane current suggest differences in neuronal signaling properties that may have important implications for integration of mechanosensory information in the deep spinal dorsal horn.

In spite of the large variability of the fraction of bursting cells reported in the subiculum (from 54 to 100%), this structure has been considered as intrinsically bursting. Here, using visually assisted whole-cell recordings and Neurobiotin labeling in vitro, we correlated the electrophysiological firing modes of subicular cells (bursting, regular-spiking and fast-spiking) with their morphological characteristics (somatic size and shape). We then examined how different morphological sampling criteria for patching affect cell classification. We found a dramatic variability in the fraction of bursting cells, which ranged from 30 to 76% depending on the sampling criteria. We discuss the implications of these findings for the notion of the subiculum as an intrinsically bursting structure.

Whole cell recordings were obtained from ventral horn neurons in spontaneously active spinal cords isolated from the chick embryo [ embryonic days 10 to 11 ( E10–E11)] to examine the post-episode depression of GABAergic transmission. Spontaneous activity occurred as recurrent, rhythmic episodes approximately 60 s in duration with 10- to 15-min quiescent inter-episode intervals. Current-clamp recording revealed that episodes were followed by a transient hyperpolarization (7 ± 1.2 mV, mean ± SE), which dissipated as a slow (0.5–1 mV/min) depolarization until the next episode. Local application of bicuculline 8 min after an episode hyperpolarized spinal neurons by 6 ± 0.8 mV and increased their input resistance by 13%, suggesting the involvement of GABAergic transmission. Gramicidin perforated-patch recordings showed that the GABAa reversal potential was above rest potential ( E GABAa = −29 ± 3 mV) and allowed estimation of the physiological intracellular [Cl−] = 50 mM. In whole cell configuration (with physiological electrode [Cl−]), two distinct types of endogenous GABAergic currents ( I GABAa) were found during the inter-episode interval. The first comprised TTX-resistant, asynchronous miniature postsynaptic currents (mPSCs), an indicator of quantal GABA release (up to 42% of total mPSCs). The second (tonic I GABAa) was complimentary to the slow membrane depolarization and may arise from persistent activation of extrasynaptic GABAa receptors. We estimate that approximately 10 postsynaptic channels are activated by a single quantum of GABA release during an mPSC and that about 30 extrasynaptic GABAa channels are required for generation of the tonic I GABAa in ventral horn neurons. We investigated the post-episode depression of I GABAa by local application of GABA or isoguvacine (100 μM, for 10–30 s) applied before and after an episode at holding potentials ( V hold) −60 mV. The amplitude of the evoked I GABA was compared after clamping the cell during the episode at one of three different V hold: −60 mV, below E GABAa resulting in Cl− efflux; −30 mV, close to E GABAa with minimal Cl− flux; and 0 mV, above E GABAa resulting in Cl− influx during the episode. The amplitude of the evoked I GABA changed according to the direction of Cl− flux during the episode: at −60 mV a 41% decrease, at −30 mV a 4% reduction, and at 0 mV a 19% increase. These post-episode changes were accompanied by shifts of E GABAa of −10, −1.2, and +7 mV, respectively. We conclude that redistribution of intracellular [Cl−] during spontaneous episodes is likely to be an important postsynaptic mechanism involved in the post-episode depression of GABAergic transmission in chick embryo spinal neurons.

The bag cells of Aplysia release egg laying hormone in sexually mature animals. Bag cells cannot sustain the long-lasting excitatory afterdischarge (AD) required for hormone release prior to sexual maturity (T.A. Nick, L.K. Kaczmarek, T.J. Carew, Ionic currents underlying developmental regulation of repetitive firing in Aplysia bag cell neurons, J. Neurosci. 1996;16:7583–7598; L.A. Fieber, Characterization of Na + and Ca 2+ currents in bag cells of sexually immature Aplysia californica , J. Exp. Biol. 1998;201:745–754). To investigate the development of bag cell excitability, whole-cell voltage-clamp experiments were executed in dissociated bag cells from four cohorts (batches) of hatchery-reared A. californica maintained at 13–15°C. K + current densities, representing the sum of at least four different outward K + currents (Nick et al., 1996), declined significantly as a function of age, beginning at least 2–3 months before sexual maturity. The K + current decreases coincided with the first appearance of Na + and Ca 2+ currents in bag cells, which occurred at ages 6–7 months. Whole cell K + currents were not decreased significantly by a cAMP analog earlier than 1 month prior to the onset of reproductive activity. The frequency of observing Na + currents in whole cell recordings was low for developmental times earlier than sexual maturity. In one winter batch, both control and PMA-treated Na + currents increased significantly with age, and PMA-treated current densities were significantly greater than controls, but the other two batches studied had significant differences in Na + current frequency only at sexual maturity. Ca 2+ currents were reliably measured in more cells than were Na + currents. The Ca 2+ current frequency increased significantly with maturity in one winter batch. Ca 2+ currents were significantly increased by phorbol ester treatment beginning 6–8 weeks before reproductive activity in the two winter batches. These observations support the hypothesis that bag cell excitability is not fully developed until shortly before sexual maturity.

Kubota, Michinori and Ikuo Taniguchi. Electrophysiological characteristics of classes of neuron in the HVc of the zebra finch. J. Neurophysiol. 80: 914–923, 1998. Whole cell recordings were made from zebra finch HVc neurons in slice preparations. Four distinct classes of neuron were found on the basis of their electrophysiological properties. The morphological characteristics of some of these neurons were also examined by intracellular injection of Lucifer yellow. Type I neurons (21 of 65 cells) had longer time-to-peak of an afterhyperpolarization following an action potential than the other classes. They exhibited both fast and time-dependent inward rectification and an initial high-frequency firing followed by a slower constant firing. Type I neurons had large somata and thick dendrites with many spines. The axons of some of the neurons in this class projected in the direction of area X of the parolfactory lobe. Type II neurons (30 of 65 cells) had a more negative resting membrane potential than the other classes. They exhibited fast inward rectification. Type II neurons could be divided into two subclasses by the absence (IIa; 22 cells) and the presence (IIb; 8 cells) of a low-threshold transient depolarization. Type IIa neurons had relatively small somata and thin, spiny dendrites. The axons of some of the neurons in this class projected in the direction of the robust nucleus of the archistriatum (RA). Type IIb neurons had relatively large somata and thick dendrites with many spines. Type III neurons (6 of 65 cells) had a shorter action-potential duration than the other classes. They exhibited prominent time-dependent inward rectification and a regular tonic firing with little or no accommodation. Type III neurons had beaded, aspiny dendrites. Type IV neurons (8 of 65 cells) had a longer action-potential duration, a much larger input resistance, and longer membrane time constant than the other classes. Type IV neurons had small somata and thin, short, sparsely spiny dendrites. The axons of some of the neurons in this class projected in the direction of the RA. These classes of neuron may play distinct roles in song production and representation in the HVc.

Visual Abstract, Sex hormone levels continuously fluctuate across the reproductive cycle, changing the activity of neuronal circuits to coordinate female behavior and reproductive capacity. The ventrolateral division of the ventromedial hypothalamus (VMHvl) contains neurons expressing receptors for sex hormones and its function is intimately linked to female sexual receptivity. However, recent findings suggest that the VMHvl is functionally heterogeneous. Here, we used whole recordings and intracellular labeling to characterize the electrophysiological and morphologic properties of individual VMHvl neurons in naturally cycling females and report the existence of multiple electrophysiological phenotypes within the VMHvl. We found that the properties of progesterone receptor expressing (PR+) neurons, but not PR– neurons, depended systematically on the neuron’s location along the anterior-posterior (AP) axis of the VMHvl and the phase within the reproductive cycle. Prominent among this, the resting membrane potential of anterior PR+ neurons decreased during the receptive phase, while the excitability of medial PR+ neurons increased during the non-receptive phase. During the receptive phase of the cycle, posterior PR+ neurons simultaneously showed an increase in dendritic complexity and a decrease in spine density. These findings reveal an extensive diversity of local rules driving structural and physiological changes in response to fluctuating levels of sex hormones, supporting the anatomic and functional subdivision of the VMHvl and its possible role in the orchestration of different aspects of female socio-sexual behavior.