In patients with damage to the primary visual cortex (V1), residual vision can guide goal-directed movements to targets in the blind field without awareness. This phenomenon has been termed ...blindsight, and its neural mechanisms are controversial. There should be visual pathways to the higher visual cortices bypassing V1, however some literature propose that the signal is mediated by the superior colliculus (SC) and pulvinar, while others claim the dorsal lateral geniculate nucleus (dLGN) transmits the signal. Here, we directly test the role of SC to ventrolateral pulvinar (vlPul) pathway in blindsight monkeys. Pharmacological inactivation of vlPul impairs visually guided saccades (VGS) in the blind field. Selective and reversible blockade of the SC-vlPul pathway by combining two viral vectors also impairs VGS. With these results we claim the SC-vlPul pathway contributes to blindsight. The discrepancy would be due to the extent of retrograde degeneration of dLGN and task used for assessment.
Midbrain dopamine neurons project densely to the striatum and form so-called dopamine synapses on medium spiny neurons (MSNs), principal neurons in the striatum. Because dopamine receptors are widely ...expressed away from dopamine synapses, it remains unclear how dopamine synapses are involved in dopaminergic transmission. Here we demonstrate that dopamine synapses are contacts formed between dopaminergic presynaptic and GABAergic postsynaptic structures. The presynaptic structure expressed tyrosine hydroxylase, vesicular monoamine transporter-2, and plasmalemmal dopamine transporter, which are essential for dopamine synthesis, vesicular filling, and recycling, but was below the detection threshold for molecules involving GABA synthesis and vesicular filling or for GABA itself. In contrast, the postsynaptic structure of dopamine synapses expressed GABAergic molecules, including postsynaptic adhesion molecule neuroligin-2, postsynaptic scaffolding molecule gephyrin, and GABAA receptor α1, without any specific clustering of dopamine receptors. Of these, neuroligin-2 promoted presynaptic differentiation in axons of midbrain dopamine neurons and striatal GABAergic neurons in culture. After neuroligin-2 knockdown in the striatum, a significant decrease of dopamine synapses coupled with a reciprocal increase of GABAergic synapses was observed on MSN dendrites. This finding suggests that neuroligin-2 controls striatal synapse formation by giving competitive advantage to heterologous dopamine synapses over conventional GABAergic synapses. Considering that MSN dendrites are preferential targets of dopamine synapses and express high levels of dopamine receptors, dopamine synapse formation may serve to increase the specificity and potency of dopaminergic modulation of striatal outputs by anchoring dopamine release sites to dopamine-sensing targets.
•Pseudotyped lentivectors with fusion glycoproteins enhance retrograde gene transfer.•The vectors have the advantage of functional manipulation of specific neural circuits.•Vector technology promotes ...the study of the mechanism underlying brain functions.•The technology offers a useful tool for gene therapy of neurological disorders.
A lentiviral vector strategy for efficient gene transfer through retrograde axonal transport provides a powerful approach for studying the neural circuit mechanisms that mediate higher level functions of the central nervous system. Pseudotyping of human immunodeficiency virus type-1 with different types of fusion glycoproteins (FuGs), which are composed of segments of rabies virus glycoprotein (RV-G) and vesicular stomatitis virus glycoprotein (VSV-G), enhances the efficiency of retrograde gene transfer in both rodent and non-human primate brains. These pseudotyped lentiviral vectors are classified into two groups, highly efficient retrograde gene transfer (HiRet) and neuron-specific retrograde gene transfer (NeuRet) vectors, based on their properties of gene transduction. Combinatorial use of the pseudotyped vectors with various molecular tools for manipulating neural circuit functions (such as the cell targeting, synaptic silencing, and optogenetic or chemogenetic approaches) enables us to control the function of specific neural circuits, thus leading to a deeper understanding of the mechanism underlying various nervous system functions.
Body fluid conditions are continuously monitored in the brain to regulate thirst and salt-appetite sensations. Angiotensin II drives both thirst and salt appetite; however, the neural mechanisms ...underlying selective water- and/or salt-intake behaviors remain unknown. Using optogenetics, we show that thirst and salt appetite are driven by distinct groups of angiotensin II receptor type 1a-positive excitatory neurons in the subfornical organ. Neurons projecting to the organum vasculosum lamina terminalis control water intake, while those projecting to the ventral part of the bed nucleus of the stria terminalis control salt intake. Thirst-driving neurons are suppressed under sodium-depleted conditions through cholecystokinin-mediated activation of GABAergic neurons. In contrast, the salt appetite-driving neurons were suppressed under dehydrated conditions through activation of another population of GABAergic neurons by Na
signals. These distinct mechanisms in the subfornical organ may underlie the selective intakes of water and/or salt and may contribute to body fluid homeostasis.
Abstract
The control of water-intake behavior is critical for life because an excessive water intake induces pathological conditions, such as hyponatremia or water intoxication. However, the brain ...mechanisms controlling water intake currently remain unclear. We previously reported that thirst-driving neurons (water neurons) in the subfornical organ (SFO) are cholecystokinin (CCK)-dependently suppressed by GABAergic interneurons under Na-depleted conditions. We herein show that CCK-producing excitatory neurons in the SFO stimulate the activity of GABAergic interneurons via CCK-B receptors. Fluorescence-microscopic Ca
2+
imaging demonstrates two distinct subpopulations in CCK-positive neurons in the SFO, which are persistently activated under hyponatremic conditions or transiently activated in response to water drinking, respectively. Optical and chemogenetic silencings of the respective types of CCK-positive neurons both significantly increase water intake under water-repleted conditions. The present study thus reveals CCK-mediated neural mechanisms in the central nervous system for the control of water-intake behaviors.
CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) ...mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20–33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as
EGFP
,
mCherry
,
Cre
, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.
Thirst and salt appetite are temporarily suppressed after water and salt ingestion, respectively, before absorption; however, the underlying neural mechanisms remain unclear. The parabrachial nucleus ...(PBN) is the relay center of ingestion signals from the digestive organs. We herein identify two distinct neuronal populations expressing cholecystokinin (Cck) mRNA in the lateral PBN that are activated in response to water and salt intake, respectively. The two Cck neurons in the dorsal-lateral compartment of the PBN project to the median preoptic nucleus and ventral part of the bed nucleus of the stria terminalis, respectively. The optogenetic stimulation of respective Cck neurons suppresses thirst or salt appetite under water- or salt-depleted conditions. The combination of optogenetics and in vivo Ca2+ imaging during ingestion reveals that both Cck neurons control GABAergic neurons in their target nuclei. These findings provide the feedback mechanisms for the suppression of thirst and salt appetite after ingestion.
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•Thirst and salt appetite are temporarily suppressed after water and salt ingestion, respectively•Two distinct subpopulations of LPBN Cck neurons are activated by water or salt ingestion•One population stimulates GABA neurons in the MnPO and the other those in the vBNST•These two pathways are involved in the suppression of thirst or salt appetite
Thirst and salt appetite are controlled based not only on body fluid conditions but also on ingestion. Matsuda et al. reveal that water and salt ingestion signals are relayed by distinct LPBN Cck neurons to GABAergic neurons in the MnPO and vBNST to suppress thirst and salt appetite, respectively.
Abstract
Social behaviour is a complex construct that is reported to include several components of social approach, interaction and recognition memory. Alzheimer’s disease (AD) is mainly ...characterized by progressive dementia and is accompanied by cognitive impairments, including a decline in social ability. The cholinergic system is a potential constituent for the neural mechanisms underlying social behaviour, and impaired social ability in AD may have a cholinergic basis. However, the involvement of cholinergic function in social behaviour has not yet been fully understood. Here, we performed a selective elimination of cholinergic cell groups in the basal forebrain in mice to examine the role of cholinergic function in social interaction and social recognition memory by using the three-chamber test. Elimination of cholinergic neurons in the medial septum (MS) and vertical diagonal band of Broca (vDB) caused impairment in social interaction, whereas ablating cholinergic neurons in the nucleus basalis magnocellularis (NBM) impaired social recognition memory. These impairments were restored by treatment with cholinesterase inhibitors, leading to cholinergic system activation. Our findings indicate distinct roles of MS/vDB and NBM cholinergic neurons in social interaction and social recognition memory, suggesting that cholinergic dysfunction may explain social ability deficits associated with AD symptoms.
A prominent feature of neocortical pyramidal cells (PCs) is their numerous projections to diverse brain areas. In layer 5 (L5) of the rat frontal cortex, there are 2 major subtypes of PCs that differ ...in their long-range axonal projections, corticopontine (CPn) cells and crossed corticostriatal (CCS) cells. The outputs of these L5 PCs can be regulated by feedback inhibition from neighboring cortical GABAergic cells. Two major subtypes of GABAergic cells are parvalbumin (PV)-positive and somatostatin (SOM)-positive cells. PV cells have a fast-spiking (FS) firing pattern, while SOM cells have a low threshold spike (LTS) and regular spiking. In this study, we found that the 2 PC subtypes in L5 selectively make recurrent connections with LTS cells. The connection patterns correlated with the morphological and physiological diversity of LTS cells. LTS cells with high input resistance (Ri) exhibited more compact dendrites and more rebound spikes than LTS cells with low Ri, which had vertically elongated dendrites. LTS subgroups differently inhibited the PC subtypes, although FS cells made nonselective connections with both projection subtypes. These results demonstrate a novel recurrent network of inhibitory and projection-specific excitatory neurons within the neocortex.
The direct cortico-motoneuronal connection is believed to be essential for the control of dexterous hand movements, such as precision grip in primates. It was reported, however, that even after ...lesion of the corticospinal tract (CST) at the C4–C5 segment, precision grip largely recovered within 1–3 mo, suggesting that the recovery depends on transmission through intercalated neurons rostral to the lesion, such as the propriospinal neurons (PNs) in the midcervical segments. To obtain direct evidence for the contribution of PNs to recovery after CST lesion, we applied a pathway-selective and reversible blocking method using double viral vectors to the PNs in six monkeys after CST lesions at C4–C5. In four monkeys that showed nearly full or partial recovery, transient blockade of PN transmission after recovery caused partial impairment of precision grip. In the other two monkeys, CST lesions were made under continuous blockade of PN transmission that outlasted the entire period of postoperative observation (3–4.5 mo). In these monkeys, precision grip recovery was not achieved. These results provide evidence for causal contribution of the PNs to recovery of hand dexterity after CST lesions; PN transmission is necessary for promoting the initial stage recovery; however, their contribution is only partial once the recovery is achieved.
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