Excessive firing of dyskinesia-associated striatal direct pathway neurons is gated by dopamine and excitatory synaptic input.

The striatum integrates dopaminergic and glutamatergic inputs to select preferred versus alternative actions. However, the precise mechanisms underlying this process remain unclear. One way to study action selection is to understand how it breaks down in pathological states. Here, we explored the cellular and synaptic mechanisms of levodopa-induced dyskinesia (LID), a complication of Parkinson's disease therapy characterized by involuntary movements. We used an activity-dependent tool (FosTRAP) in conjunction with a mouse model of LID to investigate functionally distinct subsets of striatal direct pathway medium spiny neurons (dMSNs). In vivo, levodopa differentially activates dyskinesia-associated (TRAPed) dMSNs compared to other dMSNs. We found this differential activation of TRAPed dMSNs is likely to be driven by higher dopamine receptor expression, dopamine-dependent excitability, and excitatory input from the motor cortex and thalamus. Together, these findings suggest how the intrinsic and synaptic properties of heterogeneous dMSN subpopulations integrate to support action selection.

Symmetry in Frontal But Not Motor and Somatosensory Cortical Projections.

The neocortex and striatum are topographically organized for sensory and motor functions. While sensory and motor areas are lateralized for touch and motor control, respectively, frontal areas are involved in decision-making, where lateralization of function may be less important. This study contrasted the topographic precision of cell-type-specific ipsilateral and contralateral cortical projections while varying the injection site location in transgenic mice of both sexes. While sensory cortical areas had strongly topographic outputs to the ipsilateral cortex and striatum, they were weaker and not as topographically precise to contralateral targets. The motor cortex had somewhat stronger projections but still relatively weak contralateral topography. In contrast, frontal cortical areas had high degrees of topographic similarity for both ipsilateral and contralateral projections to the cortex and striatum. Corticothalamic organization is mainly ipsilateral, with weaker, more medial contralateral projections. Corticostriatal computations might integrate input outside closed basal ganglia loops using contralateral projections, enabling the two hemispheres to act as a unit to converge on one result in motor planning and decision-making.

A distinct cortical code for socially learned threat.

Animals can learn about sources of danger while minimizing their own risk by observing how others respond to threats. However, the distinct neural mechanisms by which threats are learned through social observation (known as observational fear learning1-4 (OFL)) to generate behavioural responses specific to such threats remain poorly understood. The dorsomedial prefrontal cortex (dmPFC) performs several key functions that may underlie OFL, including processing of social information and disambiguation of threat cues5-11. Here we show that dmPFC is recruited and required for OFL in mice. Using cellular-resolution microendoscopic calcium imaging, we demonstrate that dmPFC neurons code for observational fear and do so in a manner that is distinct from direct experience. We find that dmPFC neuronal activity predicts upcoming switches between freezing and moving state elicited by threat. By combining neuronal circuit mapping, calcium imaging, electrophysiological recordings and optogenetics, we show that dmPFC projections to the midbrain periaqueductal grey (PAG) constrain observer freezing, and that amygdalar and hippocampal inputs to dmPFC opposingly modulate observer freezing. Together our findings reveal that dmPFC neurons compute a distinct code for observational fear and coordinate long-range neural circuits to select behavioural responses.

Cell-type-specific plasticity shapes neocortical dynamics for motor learning.

Neocortical spiking dynamics control aspects of behavior, yet how these dynamics emerge during motor learning remains elusive. Activity-dependent synaptic plasticity is likely a key mechanism, as it reconfigures network architectures that govern neural dynamics. Here, we examined how the mouse premotor cortex acquires its well-characterized neural dynamics that control movement timing, specifically lick timing. To probe the role of synaptic plasticity, we have genetically manipulated proteins essential for major forms of synaptic plasticity, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Cofilin, in a region and cell-type-specific manner. Transient inactivation of CaMKII in the premotor cortex blocked learning of new lick timing without affecting the execution of learned action or ongoing spiking activity. Furthermore, among the major glutamatergic neurons in the premotor cortex, CaMKII and Cofilin activity in pyramidal tract (PT) neurons, but not intratelencephalic (IT) neurons, is necessary for learning. High-density electrophysiology in the premotor cortex uncovered that neural dynamics anticipating licks are progressively shaped during learning, which explains the change in lick timing. Such reconfiguration in behaviorally relevant dynamics is impeded by CaMKII manipulation in PT neurons. Altogether, the activity of plasticity-related proteins in PT neurons plays a central role in sculpting neocortical dynamics to learn new behavior.

Symmetry in frontal but not motor and somatosensory corticocortical and corticostriatal circuitry.

Neocortex and striatum are topographically organized by cortical areas representing sensory and motor functions, where primary cortical areas are generally used as models for other cortical regions. But different cortical areas are specialized for distinct purposes, with sensory and motor areas lateralized for touch and motor control, respectively. Frontal areas are involved in decision making, where lateralization of function may be less important. This study contrasted the topographic precision of ipsilateral and contralateral projections from cortex based on the injection site location. While sensory cortical areas had strongly topographic outputs to ipsilateral cortex and striatum, they were weaker and not as topographically strong to contralateral targets. Motor cortex had somewhat stronger projections, but still relatively weak contralateral topography. In contrast, frontal cortical areas had high degrees of topographic similarity for both ipsilateral and contralateral projections to cortex and striatum. This contralateral connectivity reflects on the pathways in which corticostriatal computations might integrate input outside closed basal ganglia loops, enabling the two hemispheres to act as a single unit and converge on one result in motor planning and decision making.

Basic Neuroanatomical Methods.

This unit covers some basic procedures that are common to a wide range of neuroanatomical protocols for brain tissue. Procedures are provided for preparation of unfixed fresh brain tissue as well as for perfusion fixation of animals to obtain fixed neural tissue. A variety of methods for sectioning are described, including frozen sectioning using a cryostat or microtome and sectioning with a vibratome. The choice of sectioning method depends on how the brain has been prepared and what histochemical method is to be used. A fluorescent immunohistochemical method to localize endogenous molecules as well as induced markers such as green fluorescent protein and red fluorescent protein is also provided. Additionally, three post-sectioning procedures are described: defatting of slide-mounted sections, fluorescent Nissl staining, and thionin staining of sections. Finally, support protocols are provided, describing a method for maintaining the correct order of cut tissue, whether rostral to caudal or lateral to medial; a procedure for subbing slides with gelatin, which is necessary in some protocols in order for sections to adhere to slides; and preparation of custom 3D-printed 10- or 20-well tissue plates and trays for subsequent immunostaining. Published 2019. U.S. Government. Basic Protocol 1: Preparation of unfixed fresh-frozen brain tissue Basic Protocol 2: Perfusion fixation Basic Protocol 3: Cryostat sectioning of frozen brain tissue Basic Protocol 4: Sliding-microtome sectioning of fixed brain tissue Basic Protocol 5: Vibratome and Compresstome sectioning Support Protocol 1: Tissue collection in a 1-in-10 series Support Protocol 2: Preparation of gelatin-subbed microscope slides Support Protocol 3: Custom 3D-printed 10- and 20-well tissue plates Basic Protocol 6: Post-sectioning procedures I: Fluorescent immunohistochemical localization Basic Protocol 7: Post-sectioning procedures II: Defatting Basic Protocol 8: Post-sectioning procedures III: Nissl staining Basic Protocol 9: Post-sectioning procedures IV: Thionin staining.

Whole mouse brain reconstruction and registration to a reference atlas with standard histochemical processing of coronal sections.

Advances in molecular neuroanatomical tools have expanded the ability to map in detail connections of specific neuron subtypes in the context of behaviorally driven patterns of neuronal activity. Analysis of such data across the whole mouse brain, registered to a reference atlas, aids in understanding the functional organization of brain circuits related to behavior. A process is described to image mouse brain sections labeled with standard histochemical techniques, reconstruct those images into a whole brain image volume and register those images to the Allen Mouse Brain Common Coordinate Framework. Image analysis tools automate detection of cell bodies and quantification of axon density labeling in the structures in the annotated reference atlas. Examples of analysis are provided for mapping the axonal projections of layer-specific cortical neurons using Cre-dependent AAV vectors and for mapping inputs to such neurons using retrograde transsynaptic tracing with modified rabies viral vectors.

Author Correction: Topographic precision in sensory and motor corticostriatal projections varies across cell type and cortical area.

In the original version of this Article, support provided during initiation of the project was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include support from Karel Svoboda, members of the Svoboda lab, and members of Janelia's Vivarium staff.

Topographic precision in sensory and motor corticostriatal projections varies across cell type and cortical area.

The striatum shows general topographic organization and regional differences in behavioral functions. How corticostriatal topography differs across cortical areas and cell types to support these distinct functions is unclear. This study contrasted corticostriatal projections from two layer 5 cell types, intratelencephalic (IT-type) and pyramidal tract (PT-type) neurons, using viral vectors expressing fluorescent reporters in Cre-driver mice. Corticostriatal projections from sensory and motor cortex are somatotopic, with a decreasing topographic specificity as injection sites move from sensory to motor and frontal areas. Topographic organization differs between IT-type and PT-type neurons, including injections in the same site, with IT-type neurons having higher topographic stereotypy than PT-type neurons. Furthermore, IT-type projections from interconnected cortical areas have stronger correlations in corticostriatal targeting than PT-type projections do. As predicted by a longstanding model, corticostriatal projections of interconnected cortical areas form parallel circuits in the basal ganglia.

Whole Mouse Brain Image Reconstruction from Serial Coronal Sections Using FIJI (ImageJ).

Whole-brain reconstruction of the mouse enables comprehensive analysis of the distribution of neurochemical markers, the distribution of anterogradely labeled axonal projections or retrogradely labeled neurons projecting to a specific brain site, or the distribution of neurons displaying activity-related markers in behavioral paradigms. This unit describes a method to produce whole-brain reconstruction image sets from coronal brain sections with up to four fluorescent markers using the freely available image-processing program FIJI (ImageJ).

GENSAT BAC cre-recombinase driver lines to study the functional organization of cerebral cortical and basal ganglia circuits.

Recent development of molecular genetic techniques are rapidly advancing understanding of the functional role of brain circuits in behavior. Critical to this approach is the ability to target specific neuron populations and circuits. The collection of over 250 BAC Cre-recombinase driver lines produced by the GENSAT project provides a resource for such studies. Here we provide characterization of GENSAT BAC-Cre driver lines with expression in specific neuroanatomical pathways within the cerebral cortex and basal ganglia.

Differences between dorsal and ventral striatum in Drd1a dopamine receptor coupling of dopamine- and cAMP-regulated phosphoprotein-32 to activation of extracellular signal-regulated kinase.

Dopamine receptor signaling exhibits prominent plasticity that is important for the pathogenesis of both addictive and movement disorders. Psychoactive stimulants that activate the dopamine D(1) receptor (Drd1a) induce the rapid phosphorylation and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in neurons of the nucleus accumbens and ventral striatum. This response is known to be dependent on the phosphatase inhibitor dopamine- and cAMP-regulated phosphoprotein-32 (DARPP-32) and appears critical for the sensitization of Drd1a responses that contributes to addiction. Loss of dopamine input to the striatum, as in models of Parkinson's disease (PD), also results in a sensitization of responses to dopamine agonists that is manifest by increased activation of ERK1/2 in the dorsal striatum. Here, we test whether DARPP-32 is required for sensitization of Drd1a responses in a PD model. In the normal dorsal striatum, there is minimal Drd1a-mediated activation of ERK1/2; however, in the PD model there is robust Drd1a-mediated activation of ERK1/2. In both wild-type and DARPP-32 knock-out mice, Drd1a robustly induces pERK1/2 throughout the dopamine-depleted striatum. These findings indicate that Drd1a sensitization relevant for PD occurs by a novel mechanism that does not require DARPP-32.

Increased vulnerability of nigrostriatal terminals in DJ-1-deficient mice is mediated by the dopamine transporter.

Mutations in the gene for DJ-1 have been associated with early-onset autosomal recessive parkinsonism. Previous studies of null DJ-1 mice have shown alterations in striatal dopamine (DA) transmission with no DAergic cell loss. Here we characterize a new line of DJ-1-deficient mice. A subtle locomotor deficit was present in the absence of a change in striatal DA levels. However, increased [(3)H]-DA synaptosomal uptake and [(125)I]-RTI-121 binding were measured in null DJ-1 vs. wild-type mice. Western analyses of synaptosomes revealed significantly higher dopamine transporter (DAT) levels in pre-synaptic membrane fractions. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure exacerbated striatal DA depletion in null DJ-1 mice with no difference in DAergic nigral cell loss. Furthermore, increased 1-methyl-4-phenylpyridinium (MPP(+)) synaptosomal uptake and enhanced MPP(+) accumulation were measured in DJ-1-deficient vs. control striatum. Thus, under null DJ-1 conditions, DAT changes likely contribute to altered DA neurotransmission and enhanced sensitivity to toxins that utilize DAT for nigrostriatal entry.

D1 dopamine receptor supersensitivity in the dopamine-depleted striatum results from a switch in the regulation of ERK1/2/MAP kinase.

Dopamine effects in the striatum are mediated principally through the D1 and D2 dopamine receptor subtypes, which are segregated to the direct and indirect striatal projection neurons. After degeneration of the nigrostriatal dopamine system, direct pathway neurons display a supersensitive response to D1 dopamine receptor agonists, which is demonstrated by the induction of immediate early genes (IEGs), such as c-fos. Here we show, using analysis of receptor-mediated signal transduction, including protein phosphorylation and induction of IEGs, that D1 dopamine receptor supersensitivity is attributable to a switch to ERK1/2/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) in direct pathway neurons. Normally, in the dopamine-intact striatum, activation of ERK1/2/MAP kinase is shown to be restricted to indirect and not direct pathway neurons in response to stimulation of corticostriatal afferents. Moreover, in the dopamine-intact striatum, treatment with full D1 dopamine receptor agonists or stimulation of nigrostriatal dopaminergic afferents, both of which result in the induction of IEGs in direct striatal projection neurons, does not activate ERK1/2/MAP kinase. However, after degeneration of the nigrostriatal dopaminergic pathway, ERK1/2/MAP kinase is activated in direct pathway neurons in response to D1 dopamine receptor agonists either alone or when combined with stimulation of corticostriatal afferents. Inhibitors of MEK (MAP kinase kinase), which is responsible for phosphorylation of ERK1/2/MAP kinase, blocks D1 dopamine receptor agonist activation of ERK1/2/MAP kinase in the dopamine-depleted striatum, as well as the supersensitive induction of IEGs. These results demonstrate that dopamine input to the striatum maintains distinct forms of protein kinase-mediated gene regulation in the direct and indirect striatal projection neurons.

Reversible inhibition of CREB/ATF transcription factors in region CA1 of the dorsal hippocampus disrupts hippocampus-dependent spatial memory.

CREB is critical for long-lasting synaptic and behavioral plasticity in invertebrates. Its role in the mammalian hippocampus is less clear. We have interfered with CREB family transcription factors in region CA1 of the dorsal hippocampus. This impairs learning in the Morris water maze, which specifically requires the dorsal hippocampus, but not context conditioning, which does not. The deficit is specific to long-term memory, as shown in an object recognition task. Several forms of late-phase LTP are normal, but forskolin-induced and dopamine-regulated potentiation are disrupted. These experiments represent the first targeting of the dorsal hippocampus in genetically modified mice and confirm a role for CREB in hippocampus-dependent learning. Nevertheless, they suggest that some experimental forms of plasticity bypass the requirement for CREB.