Morpho-electric diversity of human hippocampal CA1 pyramidal neurons.

Hippocampal pyramidal neuron activity underlies episodic memory and spatial navigation. Although extensively studied in rodents, extremely little is known about human hippocampal pyramidal neurons, even though the human hippocampus underwent strong evolutionary reorganization and shows lower theta rhythm frequencies. To test whether biophysical properties of human Cornu Amonis subfield 1 (CA1) pyramidal neurons can explain observed rhythms, we map the morpho-electric properties of individual CA1 pyramidal neurons in human, non-pathological hippocampal slices from neurosurgery. Human CA1 pyramidal neurons have much larger dendritic trees than mouse CA1 pyramidal neurons, have a large number of oblique dendrites, and resonate at 2.9 Hz, optimally tuned to human theta frequencies. Morphological and biophysical properties suggest cellular diversity along a multidimensional gradient rather than discrete clustering. Across the population, dendritic architecture and a large number of oblique dendrites consistently boost memory capacity in human CA1 pyramidal neurons by an order of magnitude compared to mouse CA1 pyramidal neurons.

Integrated multimodal cell atlas of Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia in older adults. Neuropathological and imaging studies have demonstrated a progressive and stereotyped accumulation of protein aggregates, but the underlying molecular and cellular mechanisms driving AD progression and vulnerable cell populations affected by disease remain coarsely understood. The current study harnesses single cell and spatial genomics tools and knowledge from the BRAIN Initiative Cell Census Network to understand the impact of disease progression on middle temporal gyrus cell types. We used image-based quantitative neuropathology to place 84 donors spanning the spectrum of AD pathology along a continuous disease pseudoprogression score and multiomic technologies to profile single nuclei from each donor, mapping their transcriptomes, epigenomes, and spatial coordinates to a common cell type reference with unprecedented resolution. Temporal analysis of cell-type proportions indicated an early reduction of Somatostatin-expressing neuronal subtypes and a late decrease of supragranular intratelencephalic-projecting excitatory and Parvalbumin-expressing neurons, with increases in disease-associated microglial and astrocytic states. We found complex gene expression differences, ranging from global to cell type-specific effects. These effects showed different temporal patterns indicating diverse cellular perturbations as a function of disease progression. A subset of donors showed a particularly severe cellular and molecular phenotype, which correlated with steeper cognitive decline. We have created a freely available public resource to explore these data and to accelerate progress in AD research at SEA-AD.org.

Target cell-specific synaptic dynamics of excitatory to inhibitory neuron connections in supragranular layers of human neocortex.

Rodent studies have demonstrated that synaptic dynamics from excitatory to inhibitory neuron types are often dependent on the target cell type. However, these target cell-specific properties have not been well investigated in human cortex, where there are major technical challenges in reliably obtaining healthy tissue, conducting multiple patch-clamp recordings on inhibitory cell types, and identifying those cell types. Here, we take advantage of newly developed methods for human neurosurgical tissue analysis with multiple patch-clamp recordings, post-hoc fluorescent in situ hybridization (FISH), machine learning-based cell type classification and prospective GABAergic AAV-based labeling to investigate synaptic properties between pyramidal neurons and PVALB- vs. SST-positive interneurons. We find that there are robust molecular differences in synapse-associated genes between these neuron types, and that individual presynaptic pyramidal neurons evoke postsynaptic responses with heterogeneous synaptic dynamics in different postsynaptic cell types. Using molecular identification with FISH and classifiers based on transcriptomically identified PVALB neurons analyzed by Patch-seq, we find that PVALB neurons typically show depressing synaptic characteristics, whereas other interneuron types including SST-positive neurons show facilitating characteristics. Together, these data support the existence of target cell-specific synaptic properties in human cortex that are similar to rodent, thereby indicating evolutionary conservation of local circuit connectivity motifs from excitatory to inhibitory neurons and their synaptic dynamics.

Impaired dendritic spike generation in the Fragile X prefrontal cortex is due to loss of dendritic sodium channels.

Patients with Fragile X syndrome, the leading monogenetic cause of autism, suffer from impairments related to the prefrontal cortex, including working memory and attention. Synaptic inputs to the distal dendrites of layer 5 pyramidal neurons in the prefrontal cortex have a weak influence on the somatic membrane potential. To overcome this filtering, distal inputs are transformed into local dendritic Na+ spikes, which propagate to the soma and trigger action potential output. Layer 5 extratelencephalic (ET) prefrontal cortex (PFC) neurons project to the brainstem and various thalamic nuclei and are therefore well positioned to integrate task-relevant sensory signals and guide motor actions. We used current clamp and outside-out patch clamp recording to investigate dendritic spike generation in ET neurons from male wild-type and Fmr1 knockout (FX) mice. The threshold for dendritic spikes was more depolarized in FX neurons compared to wild-type. Analysis of voltage responses to simulated in vivo 'noisy' current injections showed that a larger dendritic input stimulus was required to elicit dendritic spikes in FX ET dendrites compared to wild-type. Patch clamp recordings revealed that the dendritic Na+ conductance was significantly smaller in FX ET dendrites. Taken together, our results suggest that the generation of Na+ -dependent dendritic spikes is impaired in ET neurons of the PFC in FX mice. Considering our prior findings that somatic D-type K+ and dendritic hyperpolarization-activated cyclic nucleotide-gated-channel function is reduced in ET neurons, we suggest that dendritic integration by PFC circuits is fundamentally altered in Fragile X syndrome. KEY POINTS: Dendritic spike threshold is depolarized in layer 5 prefrontal cortex neurons in Fmr1 knockout (FX) mice. Simultaneous somatic and dendritic recording with white noise current injections revealed that larger dendritic stimuli were required to elicit dendritic spikes in FX extratelencephalic (ET) neurons. Outside-out patch clamp recording revealed that dendritic sodium conductance density was lower in FX ET neurons.

Comparative cellular analysis of motor cortex in human, marmoset and mouse.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Signature morpho-electric, transcriptomic, and dendritic properties of human layer 5 neocortical pyramidal neurons.

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The unique morpho-electric properties of these neurons have been mainly described in rodents, where retrograde tracers or transgenic lines can label them. Similar labeling strategies are infeasible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology, and morphology were assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were often subtler than differences between L5 cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy but also highlight phenotypic divergence possibly related to functional specialization of human neocortex.

Scaled, high fidelity electrophysiological, morphological, and transcriptomic cell characterization.

The Patch-seq approach is a powerful variation of the patch-clamp technique that allows for the combined electrophysiological, morphological, and transcriptomic characterization of individual neurons. To generate Patch-seq datasets at scale, we identified and refined key factors that contribute to the efficient collection of high-quality data. We developed patch-clamp electrophysiology software with analysis functions specifically designed to automate acquisition with online quality control. We recognized the importance of extracting the nucleus for transcriptomic success and maximizing membrane integrity during nucleus extraction for morphology success. The protocol is generalizable to different species and brain regions, as demonstrated by capturing multimodal data from human and macaque brain slices. The protocol, analysis and acquisition software are compiled at https://githubcom/AllenInstitute/patchseqtools. This resource can be used by individual labs to generate data across diverse mammalian species and that is compatible with large publicly available Patch-seq datasets.

Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex.

Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-subclass-specific putative enhancers from across the genome to control gene expression in adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection of Parvalbumin (PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primate neocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools.

Fragile X mental retardation protein modulates somatic D-type K+ channels and action potential threshold in the mouse prefrontal cortex.

Axo-somatic K+ channels control action potential output in part by acting in concert with voltage-gated Na+ channels to set action potential threshold. Slowly inactivating, D-type K+ channels are enriched at the axo-somatic region of cortical pyramidal neurons of the prefrontal cortex, where they regulate action potential firing. We previously demonstrated that D-type K+ channels are downregulated in extratelencephalic-projecting (ET) L5 neurons in the medial prefrontal cortex (mPFC) of the Fmr1-knockout mouse model of fragile X syndrome (FX mice), resulting in a hyperpolarized action potential threshold. To test whether K+ channel alterations are regulated in a cell-autonomous manner in FXS, we used a virus-mediated approach to restore expression of fragile X mental retardation protein (FMRP) in a small population of prefrontal neurons in male FX mice. Outside-out voltage-clamp recordings revealed a higher D-type K+ conductance in FMRP-positive ET neurons compared with nearby FMRP-negative ET neurons. FMRP did not affect either rapidly inactivating A-type or noninactivating K+ conductance. ET neuron patches recorded with FMRP1-298, a truncated form of FMRP that lacks mRNA binding domains, included in the pipette solution had larger D-type K+ conductance compared with heat-inactivated controls. Viral expression of FMRP in FX mice depolarized action potential threshold to near-wild-type levels in ET neurons. These results suggest that FMRP influences the excitability of ET neurons in the mPFC by regulating somatic D-type K+ channels in a cell-autonomous, protein-protein-dependent manner.NEW & NOTEWORTHY We demonstrate that fragile X mental retardation protein (FMRP), which is absent in fragile X syndrome (FXS), regulates D-type potassium channels in prefrontal cortex L5 pyramidal neurons with subcerebral projections but not in neighboring pyramidal neurons without subcerebral projections. FMRP regulates D-type potassium channels in a protein-protein-dependent manner and rescues action potential threshold in a mouse model of FXS. These findings have implications for how changes in voltage-gated channels contribute to neurodevelopmental disorders.

Fragile X Mental Retardation Protein Bidirectionally Controls Dendritic Ih in a Cell Type-Specific Manner between Mouse Hippocampus and Prefrontal Cortex.

Channelopathies are implicated in Fragile X syndrome (FXS), yet the dysfunction of a particular ion channel varies with cell type. We previously showed that HCN channel function is elevated in CA1 dendrites of the fmr1-/y mouse model of FXS, but reduced in L5 PFC dendrites. Using male mice, we tested whether Fragile X Mental Retardation Protein (FMRPO), the protein whose absence causes FXS, differentially modulates HCN channels in CA1 versus L5 PFC dendrites. Using a combination of viral tools, intracellular peptide, and dendritic electrophysiology, we found that FMRP regulates HCN channels via a cell-autonomous protein-protein interaction. Virally expressed FMRP restored WT HCN channel-related dendritic properties in both CA1 and L5 neurons. Rapid intracellular perfusion of the non-mRNA binding N-terminal fragment, FMRP1-298, similarly restored dendritic function. In support of a protein-protein interaction, we found that FMRP associated with HCN-TRIP8b complexes in both hippocampus and PFC. Finally, voltage-clamp recordings showed that FMRP modulated Ih by regulating the number of functional dendritic HCN channels rather than individual channel properties. Together, these represent three novel findings as to the nature of the changes in dendritic function in CA1 and PFC neurons based on the presence or absence of FMRP. Moreover, our findings provide evidence that FMRP can regulate its targets in opposite directions depending upon the cellular milieu.SIGNIFICANCE STATEMENT Changes in dendritic function, and voltage-gated ion channels in particular, are increasingly the focus of neurological disorders. We, and others, previously identified cell type-specific channelopathies in a mouse of model of Fragile X syndrome. The present study shows that replacing Fragile X Mental Retardation Protein, which is absent in Fragile X syndrome, in adult CA1 and L5 PFC neurons regulates the number of functional dendritic HCN channels in a cell type-specific manner. These results suggest that Fragile X Mental Retardation Protein regulates dendritic HCN channels via a cell-autonomous protein--protein mechanism.

Transcriptomic evidence that von Economo neurons are regionally specialized extratelencephalic-projecting excitatory neurons.

von Economo neurons (VENs) are bipolar, spindle-shaped neurons restricted to layer 5 of human frontoinsula and anterior cingulate cortex that appear to be selectively vulnerable to neuropsychiatric and neurodegenerative diseases, although little is known about other VEN cellular phenotypes. Single nucleus RNA-sequencing of frontoinsula layer 5 identifies a transcriptomically-defined cell cluster that contained VENs, but also fork cells and a subset of pyramidal neurons. Cross-species alignment of this cell cluster with a well-annotated mouse classification shows strong homology to extratelencephalic (ET) excitatory neurons that project to subcerebral targets. This cluster also shows strong homology to a putative ET cluster in human temporal cortex, but with a strikingly specific regional signature. Together these results suggest that VENs are a regionally distinctive type of ET neuron. Additionally, we describe the first patch clamp recordings of VENs from neurosurgically-resected tissue that show distinctive intrinsic membrane properties relative to neighboring pyramidal neurons.

h-Channels Contribute to Divergent Intrinsic Membrane Properties of Supragranular Pyramidal Neurons in Human versus Mouse Cerebral Cortex.

Gene expression studies suggest that differential ion channel expression contributes to differences in rodent versus human neuronal physiology. We tested whether h-channels more prominently contribute to the physiological properties of human compared to mouse supragranular pyramidal neurons. Single-cell/nucleus RNA sequencing revealed ubiquitous HCN1-subunit expression in excitatory neurons in human, but not mouse, supragranular layers. Using patch-clamp recordings, we found stronger h-channel-related membrane properties in supragranular pyramidal neurons in human temporal cortex, compared to mouse supragranular pyramidal neurons in temporal association area. The magnitude of these differences depended upon cortical depth and was largest in pyramidal neurons in deep L3. Additionally, pharmacologically blocking h-channels produced a larger change in membrane properties in human compared to mouse neurons. Finally, using biophysical modeling, we provide evidence that h-channels promote the transfer of theta frequencies from dendrite-to-soma in human L3 pyramidal neurons. Thus, h-channels contribute to between-species differences in a fundamental neuronal property.

Increased transient Na+ conductance and action potential output in layer 2/3 prefrontal cortex neurons of the fmr1-/y mouse.

KEY POINTS: Layer 2/3 neurons of the prefrontal cortex display higher gain of somatic excitability, responding with a higher number of action potentials for a given stimulus, in fmr1-/y mice. In fmr1-/y L2/3 neurons, action potentials are taller, faster and narrower. Outside-out patch clamp recordings revealed that the maximum Na+ conductance density is higher in fmr1-/y L2/3 neurons. Measurements of three biophysically distinct K+ currents revealed a depolarizing shift in the activation of a rapidly inactivating (A-type) K+ conductance. Realistic neuronal simulations of the biophysical observations recapitulated the elevated action potential and repetitive firing phenotype. ABSTRACT: Fragile X syndrome is the most common form of inherited mental impairment and autism. The prefrontal cortex is responsible for higher order cognitive processing, and prefrontal dysfunction is believed to underlie many of the cognitive and behavioural phenotypes associated with fragile X syndrome. We recently demonstrated that somatic and dendritic excitability of layer (L) 5 pyramidal neurons in the prefrontal cortex of the fmr1-/y mouse is significantly altered due to changes in several voltage-gated ion channels. In addition to L5 pyramidal neurons, L2/3 pyramidal neurons play an important role in prefrontal circuitry, integrating inputs from both lower brain regions and the contralateral cortex. Using whole-cell current clamp recording, we found that L2/3 pyramidal neurons in prefrontal cortex of fmr1-/y mouse fired more action potentials for a given stimulus compared with wild-type neurons. In addition, action potentials in fmr1-/y neurons were significantly larger, faster and narrower. Voltage clamp of outside-out patches from L2/3 neurons revealed that the transient Na+ current was significantly larger in fmr1-/y neurons. Furthermore, the activation curve of somatic A-type K+ current was depolarized. Realistic conductance-based simulations revealed that these biophysical changes in Na+ and K+ channel function could reliably reproduce the observed increase in action potential firing and altered action potential waveform. These results, in conjunction with our prior findings on L5 neurons, suggest that principal neurons in the circuitry of the medial prefrontal cortex are altered in distinct ways in the fmr1-/y mouse and may contribute to dysfunctional prefrontal cortex processing in fragile X syndrome.

Systems-based analysis of dendritic nonlinearities reveals temporal feature extraction in mouse L5 cortical neurons.

What do dendritic nonlinearities tell a neuron about signals injected into the dendrite? Linear and nonlinear dendritic components affect how time-varying inputs are transformed into action potentials (APs), but the relative contribution of each component is unclear. We developed a novel systems-identification approach to isolate the nonlinear response of layer 5 pyramidal neuron dendrites in mouse prefrontal cortex in response to dendritic current injections. We then quantified the nonlinear component and its effect on the soma, using functional models composed of linear filters and static nonlinearities. Both noise and waveform current injections revealed linear and nonlinear components in the dendritic response. The nonlinear component consisted of fast Na+ spikes that varied in amplitude 10-fold in a single neuron. A functional model reproduced the timing and amplitude of the dendritic spikes and revealed that they were selective to a preferred input dynamic (~4.5 ms rise time). The selectivity of the dendritic spikes became wider in the presence of additive noise, which was also predicted by the functional model. A second functional model revealed that the dendritic spikes were weakly boosted before being linearly integrated at the soma. For both our noise and waveform dendritic input, somatic APs were dependent on the somatic integration of the stimulus, followed a subset of large dendritic spikes, and were selective to the same input dynamics preferred by the dendrites. Our results suggest that the amplitude of fast dendritic spikes conveys information about high-frequency features in the dendritic input, which is then combined with low-frequency somatic integration.NEW & NOTEWORTHY The nonlinear response of layer 5 mouse pyramidal dendrites was isolated with a novel systems-based approach. In response to dendritic current injections, the nonlinear component contained mostly fast, variable-amplitude, Na+ spikes. A functional model accounted for the timing and amplitude of the dendritic spikes and revealed that dendritic spikes are selective to a preferred input dynamic, which was verified experimentally. Thus, fast dendritic nonlinearities behave as high-frequency feature detectors that influence somatic action potentials.

Cell-Type Specific Channelopathies in the Prefrontal Cortex of the fmr1-/y Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is caused by transcriptional silencing of the fmr1 gene resulting in the loss of fragile X mental retardation protein (FMRP) expression. FXS patients display several behavioral phenotypes associated with prefrontal cortex (PFC) dysfunction. Voltage-gated ion channels, some of which are regulated by FMRP, heavily influence PFC neuron function. Although there is evidence for brain region-specific alterations to the function a single type of ion channel in FXS, it is unclear whether subtypes of principal neurons within a brain region are affected uniformly. We tested for alterations to ion channels critical in regulating neural excitability in two subtypes of prefrontal L5 pyramidal neurons. Using somatic and dendritic patch-clamp recordings, we provide evidence that the functional expression of h-channels (Ih) is down-regulated, whereas A-type K(+) channel function is up-regulated in pyramidal tract-projecting (PT) neurons in the fmr1-/y mouse PFC. This is the opposite pattern of results from published findings from hippocampus where Ih is up-regulated and A-type K(+) channel function is down-regulated. Additionally, we find that somatic Kv1-mediated current is down-regulated, resulting in increased excitability of fmr1-/y PT neurons. Importantly, these h- and K(+) channel differences do not extend to neighboring intratelencephalic-projecting neurons. Thus, the absence of FMRP has divergent effects on the function of individual types of ion channels not only between brain regions, but also variable effects across cell types within the same brain region. Given the importance of ion channels in regulating neural circuits, these results suggest cell-type-specific phenotypes for the disease.

Species-specific differences in the medial prefrontal projections to the pons between rat and rabbit.

The medial prefrontal cortex (mPFC) of both rats and rabbits has been shown to support trace eyeblink conditioning, presumably by providing an input to the cerebellum via the pons that bridges the temporal gap between conditioning stimuli. The pons of rats and rabbits, however, shows divergence in gross anatomical organization, leaving open the question of whether the topography of prefrontal inputs to the pons is similar in rats and rabbits. To investigate this question, we injected anterograde tracer into the mPFC of rats and rabbits to visualize and map in 3D the distribution of labeled terminals in the pons. Effective mPFC injections showed labeled axons in the ipsilateral descending pyramidal tract in both species. In rats, discrete clusters of densely labeled terminals were observed primarily in the rostromedial pons. Clusters of labeled terminals were also observed contralateral to mPFC injection sites in rats, appearing as a less dense "mirror-image" of ipsilateral labeling. In rabbits, mPFC labeled corticopontine terminals were absent in the rostral pons, and instead were restricted to the intermediate pons. The densest terminal fields were typically observed in association with the ipsilateral pyramidal tract as it descended ventromedially through the rabbit pons. No contralateral terminal labeling was observed for any injections made in the rabbit mPFC. The results suggest the possibility that mPFC inputs to the pons may be integrated with different sources of cortical inputs between rats and rabbits. The resulting implications for mPFC or pons manipulations for studies of trace eyeblink in each species are discussed.

Dendritic generation of mGluR-mediated slow afterdepolarization in layer 5 neurons of prefrontal cortex.

Many prefrontal cortex (PFC)-dependent tasks require individual neurons to fire persistently in response to brief stimuli. Persistent activity is proposed to involve changes in intrinsic properties, resulting in an increased sensitivity to inputs. The dendrite is particularly relevant to this hypothesis because it receives the majority of synaptic inputs and is enriched for conductances implicated in persistent firing. We provide evidence that dendritic conductances contribute to persistent activity-related changes in intrinsic properties. The effects of Group 1 metabotropic glutamate receptor (mGluR) activation on persistent activity-related properties were tested in two classes of rat L5 neurons with distinct membrane properties: those projecting to the pons (CPn) and those projecting across the commissure to the contralateral cortex (COM). mGluR activation produced long-term changes in the subthreshold properties of CPn, but not COM neurons. These changes were indicative of a decrease in hyperpolarization-activated cation nonselective current (I(h)) at the soma and dendrite. mGluR activation also transiently increased the amplitude of the postburst slow afterdepolarization potential (sADP) at the soma of both neuron types. Interestingly, the sADP occurred along the extent of the apical dendrite in CPn and COM neurons. Simultaneous somatic/dendritic recordings revealed that the dendritic sADP does not result solely from passive propagation of the somatic sADP. Focal mGluR activation in L5, near the soma or at the border of L1/L2, near the tuft, generates a local sADP. This dendritic depolarization may act synergistically with synaptic input to regulate mnemonic activity in PFC.

Multiple sites of extinction for a single learned response.

Most learned responses can be diminished by extinction, a process that can be engaged when a conditioned stimulus (CS) is presented but not reinforced. We present evidence that plasticity in at least two brain regions can mediate extinction of responses produced by trace eyelid conditioning, where the CS and the reinforcing stimulus are separated by a stimulus-free interval. We observed individual differences in the effects of blocking extinction mechanisms in the cerebellum, the structure that, along with several forebrain structures, mediates acquisition of trace eyelid responses; in some rabbits extinction was prevented, whereas in others it was largely unaffected. We also show that cerebellar mechanisms can mediate extinction when noncerebellar mechanisms are bypassed. Together, these observations indicate that trace eyelid responses can be extinguished via processes operating at more than one site, one in the cerebellum and one upstream in forebrain. The relative contributions of these sites may vary from animal to animal and situation to situation.

A subtraction mechanism of temporal coding in cerebellar cortex.

The temporally specific learning displayed by the cerebellum facilitates mechanistic analysis of neural timing and temporal coding. We report evidence for a subtraction-like mechanism of temporal coding in cerebellar cortex in which activity in a subset of granule cells specifically codes the interval between the offset of two mossy fiber inputs. In a large-scale cerebellar simulation, cessation of one of two ongoing mossy fiber inputs produces a robust temporal code in the population of granule cells. This activity supports simulation learning in response to temporal patterns of stimuli, even when those same stimuli do not support learning when presented individually. Using stimulation of mossy fiber inputs to the cerebellum as training stimuli in rabbits, we confirmed these unusual predictions in a cerebellum-dependent form of learning. Analysis of the simulations reveals a specific working hypothesis for this temporal subtraction process that involves interactions between granule cells and the inhibitory Golgi cells. The results suggest how feedforward inhibition, such as that present in the cerebellar cortex, can contribute to temporal coding.