The basal forebrain (BF) receives afferents from brainstem ascending pathways, which has been implicated first by Moruzzi and Magoun (1949) to induce forebrain activation and cortical arousal/waking behavior; however, it is very little known about how brainstem inhibitory inputs affect cholinergic functions. In the current study, glycine, a major inhibitory neurotransmitter of brainstem neurons, and gliotransmitter of local glial cells, was tested for potential interaction with BF cholinergic (BFC) neurons in male mice. In the BF, glycine receptor α subunit-immunoreactive (IR) sites were localized in choline acetyltransferase (ChAT)-IR neurons. The effect of glycine on BFC neurons was demonstrated by bicuculline-resistant, strychnine-sensitive spontaneous IPSCs (sIPSCs; 0.81 ± 0.25 × 10-1 Hz) recorded in whole-cell conditions. Potential neuronal as well as glial sources of glycine were indicated in the extracellular space of cholinergic neurons by glycine transporter type 1 (GLYT1)- and GLYT2-IR processes found in apposition to ChAT-IR cells. Ultrastructural analyses identified synapses of GLYT2-positive axon terminals on ChAT-IR neurons, as well as GLYT1-positive astroglial processes, which were localized in the vicinity of synapses of ChAT-IR neurons. The brainstem raphe magnus was determined to be a major source of glycinergic axons traced retrogradely from the BF. Our results indicate a direct effect of glycine on BFC neurons. Furthermore, the presence of high levels of plasma membrane glycine transporters in the vicinity of cholinergic neurons suggests a tight control of extracellular glycine in the BF.SIGNIFICANCE STATEMENT Basal forebrain cholinergic (BFC) neurons receive various activating inputs from specific brainstem areas and channel this information to the cortex via multiple projections. So far, very little is known about inhibitory brainstem afferents to the BF. The current study established glycine as a major regulator of BFC neurons by (1) identifying glycinergic neurons in the brainstem projecting to the BF, (2) showing glycine receptor α subunit-immunoreactive (IR) sites in choline acetyltransferase (ChAT)-IR neurons, (3) demonstrating glycine transporter type 2 (GLYT2)-positive axon terminals synapsing on ChAT-IR neurons, and (4) localizing GLYT1-positive astroglial processes in the vicinity of synapses of ChAT-IR neurons. The effect of glycine on BFC neurons was demonstrated by bicuculline-resistant, strychnine-sensitive spontaneous IPSCs recorded in whole-cell conditions.

The basal forebrain (BF) plays an important role in the control of cortical activation and attention. Understanding the modulation of BF neuronal activity is a prerequisite to treat disorders of cortical activation involving BF dysfunction, such as Alzheimer's disease. Here we reveal the interaction between cholinergic neurons and cortically projecting BF GABAergic neurons using immunohistochemistry and whole-cell recordings in vitro. In GAD67-GFP knock-in mice, BF cholinergic (choline acetyltransferase-positive) neurons were intermingled with GABAergic (GFP(+)) neurons. Immunohistochemistry for the vesicular acetylcholine transporter showed that cholinergic fibers apposed putative cortically projecting GABAergic neurons containing parvalbumin (PV). In coronal BF slices from GAD67-GFP knock-in or PV-tdTomato mice, pharmacological activation of cholinergic receptors with bath application of carbachol increased the firing rate of large (>20 μm diameter) BF GFP(+) and PV (tdTomato+) neurons, which exhibited the intrinsic membrane properties of cortically projecting neurons. The excitatory effect of carbachol was blocked by antagonists of M1 and M3 muscarinic receptors in two subpopulations of BF GABAergic neurons [large hyperpolarization-activated cation current (Ih) and small Ih, respectively]. Ion substitution experiments and reversal potential measurements suggested that the carbachol-induced inward current was mediated mainly by sodium-permeable cation channels. Carbachol also increased the frequency of spontaneous excitatory and inhibitory synaptic currents. Furthermore, optogenetic stimulation of cholinergic neurons/fibers caused a mecamylamine- and atropine-sensitive inward current in putative GABAergic neurons. Thus, cortically projecting, BF GABAergic/PV neurons are excited by neighboring BF and/or brainstem cholinergic neurons. Loss of cholinergic neurons in Alzheimer's disease may impair cortical activation, in part, through disfacilitation of BF cortically projecting GABAergic/PV neurons.

Basal forebrain (BF) projections to the hippocampus and cortex are anatomically positioned to influence a broad range of cognitive capacities that are known to decline in normal aging, including executive function and memory. Although a long history of research on neurocognitive aging has focused on the role of the cholinergic basal forebrain system, intermingled GABAergic cells are numerically as prominent and well positioned to regulate the activity of their cortical projection targets, including the hippocampus and prefrontal cortex. The effects of aging on noncholinergic BF neurons in primates, however, are largely unknown. In this study, we conducted quantitative morphometric analyses in brains from young adult (6 females, 2 males) and aged (11 females, 5 males) rhesus monkeys (Macaca mulatta) that displayed significant impairment on standard tests that require the prefrontal cortex and hippocampus. Cholinergic (ChAT+) and GABAergic (GAD67+) neurons were quantified through the full rostrocaudal extent of the BF. Total BF immunopositive neuron number (ChAT+ plus GAD67+) was significantly lower in aged monkeys compared with young, largely because of fewer GAD67+ cells. Additionally, GAD67+ neuron volume was greater selectively in aged monkeys without cognitive impairment compared with young monkeys. These findings indicate that the GABAergic component of the primate BF is disproportionally vulnerable to aging, implying a loss of inhibitory drive to cortical circuitry. Moreover, adaptive reorganization of the GABAergic circuitry may contribute to successful neurocognitive outcomes.SIGNIFICANCE STATEMENT A long history of research has confirmed the role of the basal forebrain in cognitive aging. The majority of that work has focused on BF cholinergic neurons that innervate the cortical mantle. Codistributed BF GABAergic populations are also well positioned to influence cognitive function, yet little is known about this prominent neuronal population in the aged brain. In this unprecedented quantitative comparison of both cholinergic and GABAergic BF neurons in young and aged rhesus macaques, we found that neuron number is significantly reduced in the aged BF compared with young, and that this reduction is disproportionately because of a loss of GABAergic neurons. Together, our findings encourage a new perspective on the functional organization of the primate BF in neurocognitive aging.

General anesthesia shares many similarities with natural sleep in behavior and electroencephalogram (EEG) patterns. The latest evidence suggests that general anesthesia and sleep-wake behavior may share overlapping neural substrates. The GABAergic neurons in the basal forebrain (BF) have recently been demonstrated to play a key role in controlling wakefulness. It was hypothesized that BF GABAergic neurons may participate in the regulation of general anesthesia. Here, using in vivo fiber photometry, we found that the activity of BF GABAergic neurons was generally inhibited during isoflurane anesthesia, having obviously decreased during the induction of anesthesia and being gradually restored during the emergence from anesthesia, in Vgat-Cre mice of both sexes. Activation of BF GABAergic neurons with chemogenetic and optogenetic approaches decreased sensitivity to isoflurane, delayed induction, and accelerated emergence from isoflurane anesthesia. Optogenetic activation of BF GABAergic neurons decreased EEG δ power and the burst suppression ratio (BSR) during 0.8% and 1.4% isoflurane anesthesia, respectively. Similar to the effects of activating BF GABAergic cell bodies, photostimulation of BF GABAergic terminals in the thalamic reticular nucleus (TRN) also strongly promoted cortical activation and behavioral emergence from isoflurane anesthesia. Collectively, these results showed that the GABAergic BF is a key neural substrate for general anesthesia regulation that facilitates behavioral and cortical emergence from general anesthesia via the GABAergic BF-TRN pathway. Our findings may provide a new target for attenuating the depth of anesthesia and accelerating emergence from general anesthesia.SIGNIFICANCE STATEMENT The basal forebrain (BF) is a key brain region controlling sleep-wake behavior. Activation of GABAergic neurons in the BF potently promotes behavioral arousal and cortical activity. Recently, many sleep-wake-related brain structures have been reported to participate in the regulation of general anesthesia. However, it is still unclear what role BF GABAergic neurons play in general anesthesia. In this study, we aim to reveal the role of BF GABAergic neurons in behavioral and cortical emergence from isoflurane anesthesia and elucidate the underlying neural pathways. Understanding the specific role of BF GABAergic neurons in isoflurane anesthesia would improve our understanding of the mechanisms of general anesthesia and may provide a new strategy for accelerating emergence from general anesthesia.

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, we showed that mice (both females and males) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1+/-) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1+/- mice showed a robust decrease in methamphetamine-induced dopamine release in the NAc with no change in baseline extracellular dopamine, striatal whole-tissue dopamine, dopamine transporter protein, dopamine uptake, or striatal methamphetamine and amphetamine metabolite levels. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for TH did not reveal any major changes in staining intensity, cell number, or forebrain puncta counts. Surprisingly, there was a twofold increase in hnRNP H protein in the striatal synaptosome of H1+/- mice with no change in whole-tissue levels. To gain insight into the mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min after methamphetamine administration in H1+/- mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in WT mice showed a rapid increase. We conclude that H1+/- decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.SIGNIFICANCE STATEMENT Methamphetamine dependence is a significant public health concern with no FDA-approved treatment. We discovered a role for the RNA binding protein hnRNP H in methamphetamine reward and reinforcement. Hnrnph1 mutation also blunted methamphetamine-induced dopamine release in the NAc, a key neurochemical event contributing to methamphetamine addiction liability. Finally, Hnrnph1 mutants showed a marked increase in basal level of synaptosomal hnRNP H and mitochondrial proteins that decreased in response to methamphetamine, whereas WT mice showed a methamphetamine-induced increase in synaptosomal mitochondrial proteins. Thus, we identified a potential role for hnRNP H in basal and dynamic mitochondrial function that informs methamphetamine-induced cellular adaptations associated with reduced addiction liability.

Wake neurons in the basal forebrain and brainstem provide critical inputs to optimize alertness and attention. These neurons, however, evidence heightened vulnerability to a diverse array of metabolic challenges, including aging. SIRT1 is an nicotinamide adenine dinucleotide responsive deacetylase serving diverse adaptive responses to metabolic challenges, yet this metabolic rheostat may be downregulated under conditions of significant oxidative stress. We hypothesized that SIRT1 might serve as a critical neuroprotectant for wake neurons in young animals but that this protectant would be lost upon aging, rendering the neurons more vulnerable to metabolic insults. In this collection of studies, we first established the presence of nuclear SIRT1 in wake neurons throughout the forebrain and brainstem. Supporting functional and behavioral roles for SIRT1 in wake-active neurons, transgenic whole animal, and conditional loss of brain SIRT1 in the adult mouse impart selective impairments in wakefulness, without disrupting non-rapid eye movement or rapid eye movement sleep. Populations of wake neurons, including the orexinergic, locus ceruleus, mesopontine cholinergic, and dopaminergic wake neurons, evidence loss of dendrites and neurotransmitter synthesis enzymes and develop accelerated accumulation of lipofuscin, consistent with a senescence-like phenotype in wake neurons. Normal aging results in a progressive loss of SIRT1 in wake-active neurons, temporally coinciding with lipofuscin accumulation. SIRT1 is a critical age-sensitive neuroprotectant for wake neurons, and its deficiency results in impaired wakefulness.

Immunohistochemical localization of choline acetyltransferase (ChAT) in cholinergic neurons has been difficult to achieve because of problems encountered in producing specific antisera. Here we describe the production and characterization of several distinct monoclonal antibodies to ChAT. Each of the monoclonal antibodies exhibits one of three general patterns of cross-species reactions; one pattern shows reactivity limited mainly to bovine ChAT, a second pattern shows reactivity only to ChAT from higher mammals including humans, and the third pattern shows reactivity to ChAT from all mammals tested. The antibodies bound specifically to two closely related bovine proteins of 68,000 and 70,000 daltons using the Western blotting technique. One of the antibodies was used to localize immunohistochemically known cholinergic structures in the rat brain, including motor neurons, basal forebrain neurons, and neostriatal neurons.

Integrin-linked kinase (Ilk) is a scaffold and kinase that links integrin receptors to the actin cytoskeleton and to signaling pathways involved in cell adhesion, migration, and extracellular matrix deposition. Targeted deletion of Ilk from embryonic mouse dorsal forebrain neuroepithelium results in severe cortical lamination defects resembling cobblestone (type II) lissencephaly. Defects in adult mutants include neuronal invasion of the marginal zone, downward displacement of marginal zone components, fusion of the cerebral hemispheres, and scalloping of the dentate gyrus. These lesions are associated with abundant astrogliosis and widespread fragmentation of the basal lamina at the cortical surface. During cortical development, neuronal ectopias are associated with severe disorganization of radial glial processes and displacement of Cajal-Retzius cells. Lesions are not seen when Ilk is specifically deleted from embryonic neurons. Interestingly, targeted Ilk deletion has no effect on proliferation or survival of cortical cells or on phosphorylation of two Ilk substrates, Pkb/Akt and Gsk-3beta, suggesting that Ilk does not regulate cortical lamination via these enzymes. Instead, Ilk acts in vivo as a major intracellular mediator of integrin-dependent basal lamina formation. This study demonstrates a critical role for Ilk in cortical lamination and suggests that Ilk-associated pathways are involved in the pathogenesis of cobblestone lissencephalies.

The presence of novel or degraded communication sounds likely results in activation of basal forebrain cholinergic neurons increasing release of ACh onto presynaptic and postsynaptic nAChRs in primary auditory cortex (A1). nAChR subtypes include high-affinity heteromeric nAChRs commonly composed of α4 and β2 subunits and low-affinity homomeric nAChRs composed of α7 subunits. In young male FBN rats, we detail the following: (1) the distribution/expression of nAChR subunit transcripts in excitatory (VGluT1) and inhibitory (VGAT) neurons across A1 layers; (2) heteromeric nAChR binding across A1 layers; and (3) nAChR excitability in A1 layer (L) 5 cells. In aged rats, we detailed the impact of aging on A1 nAChR subunit expression across layers, heteromeric nAChR receptor binding, and nAChR excitability of A1 L5 cells. A majority of A1 cells coexpressed transcripts for β2 and α4 with or without α7, while dispersed subpopulations expressed β2 and α7 or α7 alone. nAChR subunit transcripts were expressed in young excitatory and inhibitory neurons across L2-L6. Transcript abundance varied across layers, and was highest for β2 and α4. Significant age-related decreases in nAChR subunit transcript expression (message) and receptor binding (protein) were observed in L2-6, most pronounced in infragranular layers. In vitro patch-clamp recordings from L5B pyramidal output neurons showed age-related nAChR subunit-selective reductions in postsynaptic responses to ACh. Age-related losses of nAChR subunits likely impact ways in which A1 neurons respond to ACh release. While the elderly require additional resources to disambiguate degraded speech codes, resources mediated by nAChRs may be compromised with aging.SIGNIFICANCE STATEMENT When attention is required, cholinergic basal forebrain neurons may trigger increased release of ACh onto auditory neurons in primary auditory cortex (A1). Laminar and phenotypic differences in neuronal nAChR expression determine ways in which A1 neurons respond to release of ACh in challenging acoustic environments. This study detailed the distribution and expression of nAChR subunit transcript and protein across A1 layers in young and aged rats. Results showed a differential distribution of nAChR subunits across A1 layers. Age-related decreases in transcript/protein expression were reflected in age-related subunit specific functional loss of nAChR signaling to ACh application in A1 layer 5. Together, these findings could reflect the age-related decline in selective attention observed in the elderly.

Rhesus monkeys (Macaca mulatta) reared during the first year of life without social contact develop persistent stereotyped movements, self-directed behaviors, and psychosocial abnormalities, but neurobiological mechanisms underlying the behaviors of socially deprived (SD) monkeys are unknown. Monkeys were reared in total social deprivation for the first 9 months of life; control monkeys were reared socially (SR) with mothers and peers. Subjects were killed at 19-24 yr of age. Because the behaviors of SD monkeys are reminiscent of changes in striatal or amygdalar function, we used immunocytochemistry for substance P (SP), leucine-enkephalin (LENK), somatostatin, calbindin, and tyrosine hydroxylase (TH) to evaluate qualitatively and quantitatively patterns of neurotransmitter marker immunoreactivity within subcortical regions. In SD monkeys, the chemoarchitecture of the striatum was altered. Neuronal cell bodies and processes immunoreactive for SP and LENK were depleted markedly in patch (striosome) and matrix regions of the caudate nucleus and putamen; the average density of SP-immunoreactive neurons was reduced 58% relative to SR monkeys. Calbindin and TH immunoreactivities were diminished in the matrix of caudate and putamen of SD monkeys. TH-immunoreactive neurons, but not cresyl violet-stained neurons, in the substantia nigra pars compacta were decreased (43%) in SD monkeys. Peptide-immunoreactive terminals were reduced in the globus pallidus and substantia nigra in SD monkeys. The nucleus accumbens was the least affected of striatal regions. Striatal somatostatin immunoreactivity wa qualitatively and quantitatively similar in SD and SR monkeys. Several regions, for example, bed nucleus of the stria terminalis, amygdala, and basal forebrain magnocellular complex, that were in the same sections and are enriched in these markers did not appear altered in SD monkeys, suggesting a regional specificity for vulnerability. The altered chemoarchitecture of some basal ganglia regions in adult monkeys that experienced social deprivation as infants suggests that the postnatal maturation of neurotransmitter phenotypes in some structures is influenced by social environment. Abnormal motor and psychosocial behaviors resulting from this form of social/sensory deprivation may result from alterations in peptidergic and dopaminergic systems within the basal ganglia.

The projection neurons of the striatum, the principal nucleus of the basal ganglia, belong to one of the following two major pathways: the striatopallidal (indirect) pathway or the striatonigral (direct) pathway. Striatonigral axons project long distances and encounter ascending tracts (thalamocortical) while coursing alongside descending tracts (corticofugal) as they extend through the internal capsule and cerebral peduncle. These observations suggest that striatal circuitry may help to guide their trajectories. To investigate the developmental contributions of striatonigral axons to internal capsule formation, we have made use of Sox8-EGFP (striatal direct pathway) and Fezf2-TdTomato (corticofugal pathway) BAC transgenic reporter mice in combination with immunohistochemical markers to trace these axonal pathways throughout development. We show that striatonigral axons pioneer the internal capsule and cerebral peduncle and are temporally and spatially well positioned to provide guidance for corticofugal and thalamocortical axons. Using Isl1 conditional knock-out (cKO) mice, which exhibit disrupted striatonigral axon outgrowth, we observe both corticofugal and thalamocortical axon defects with either ventral forebrain- or telencephalon-specific Isl1 inactivation, despite Isl1 not being expressed in either cortical or thalamic projection neurons. Striatonigral axon defects can thus disrupt internal capsule formation. Our genome-wide transcriptomic analysis in Isl1 cKOs reveals changes in gene expression relevant to cell adhesion, growth cone dynamics, and extracellular matrix composition, suggesting potential mechanisms by which the striatonigral pathway exerts this guidance role. Together, our data support a novel pioneering role for the striatal direct pathway in the correct assembly of the ascending and descending axon tracts within the internal capsule and cerebral peduncle.SIGNIFICANCE STATEMENT The basal ganglia are a group of subcortical nuclei with established roles in the coordination of voluntary motor programs, aspects of cognition, and the selection of appropriate social behaviors. Hence, disruptions in basal ganglia connectivity have been implicated in the motor, cognitive, and social dysfunction characterizing common neurodevelopmental disorders such as attention-deficit/hyperactivity disorder, autism spectrum disorder, obsessive-compulsive disorder, and tic disorder. Here, we identified a novel role for the striatonigral (direct) pathway in pioneering the internal capsule and cerebral peduncle, and in guiding axons extending to and from the cortex. Our findings suggest that the abnormal development of basal ganglia circuits can drive secondary internal capsule defects and thereby may contribute to the pathology of these disorders.

We have previously shown that neurons in the basal forebrain colocalize the neurotrophin receptor p75NGFR and estrogen receptors. The present study was designed to examine (1) if neural neurotrophin targets respond to estrogen as a general phenotypic feature and (2) if NGF receptor mRNAs are regulated by estrogen, using a prototypical target of NGF, the dorsal root ganglion (DRG) (sensory) neuron. We demonstrate, for the first time, the presence of estrogen receptor mRNA and protein (binding sites) in adult female rat DRG. Moreover, estrogen receptor mRNA expression, while present in DRG neurons from both the ovariectomized (OVX; estrogen deficient) and intact female rat, was downregulated, as in the adult CNS, during proestrus (high estrogen levels) and in OVX animals replaced with proestrus levels of estrogen, as compared to OVX controls. In contrast, although the mRNAs for the NGF receptors p75NGFR and trkA were also expressed in DRG neurons from OVX and intact animals, expression of both NGF receptor mRNAs was upregulated in sensory neurons during proestrus, as compared to the OVX condition. Estrogen replacement, on the other hand, resulted in a transient downregulation of p75NGFR mRNA and a time-dependent upregulation of trkA mRNA. Estrogen regulation of NGF receptor mRNA in adult peripheral neural targets of the neurotrophins supports the hypothesis that estrogen may regulate neuronal sensitivity to neurotrophins such as NGF and may be an important mediator of neurotrophin actions in normal neural function and following neural trauma.

Coiled-coil and C2 domain containing 1A (CC2D1A) is an evolutionarily conserved protein, originally identified as a nuclear factor-κB activator through a large-scale screen of human genes. Mutations in the human Cc2d1a gene result in autosomal recessive nonsyndromic intellectual disability. It remains unclear, however, how Cc2d1a mutation leads to alterations in brain function. Here, we have taken advantage of Cre/loxP recombinase-based strategy to conditionally delete Cc2d1a exclusively from excitatory neurons of male mouse forebrain to examine its role in hippocampal synaptic plasticity and cognitive function. We confirmed the expression of CC2D1A protein and mRNA in the mouse hippocampus. Double immunofluorescence staining showed that CC2D1A is expressed in both excitatory and inhibitory neurons of the adult hippocampus. Conditional deletion of Cc2d1a (cKO) from excitatory neurons leads to impaired performance in object location memory test and altered anxiety-like behavior. Consistently, cKO mice displayed a deficit in the maintenance of LTP in the CA1 region of hippocampal slices. Cc2d1a deletion also resulted in decreased complexity of apical and basal dendritic arbors of CA1 pyramidal neurons. An enhanced basal Rac1 activity was observed following Cc2d1a deletion, and this enhancement was mediated by reduced SUMO-specific protease 1 (SENP1) and SENP3 expression, thus increasing the amount of Rac1 SUMOylation. Furthermore, partial blockade of Rac1 activity rescued impairments in LTP and object location memory performance in cKO mice. Together, our results implicate Rac1 hyperactivity in synaptic plasticity and cognitive deficits observed in Cc2d1a cKO mice and reveal a novel role for CC2D1A in regulating hippocampal synaptic function.SIGNIFICANCE STATEMENT CC2D1A is abundantly expressed in the brain, but there is little known about its physiological function. Taking advantage of Cc2d1a cKO mice, the present study highlights the importance of CC2D1A in the maintenance of LTP at Schaffer collateral-CA1 synapses and the formation of hippocampus-dependent long-term object location memory. Our findings establish a critical link between elevated Rac1 activity, structural and synaptic plasticity alterations, and cognitive impairment caused by Cc2d1a deletion. Moreover, partial blockade of Rac1 activity rescues synaptic plasticity and memory deficits in Cc2d1a cKO mice. Such insights may have implications for the utility of Rac1 inhibitors in the treatment of intellectual disability caused by Cc2d1a mutations in human patients.

Oxytocin (OXT) receptors (OXTRs) are prominently expressed in hippocampal CA2 and CA3 pyramidal neurons, but little is known about its physiological function. As the functional necessity of hippocampal CA2 for social memory processing, we tested whether CA2 OXTRs may contribute to long-term social recognition memory (SRM) formation. Here, we found that conditional deletion of Oxtr from forebrain (Oxtr-/-) or CA2/CA3a-restricted excitatory neurons in adult male mice impaired the persistence of long-term SRM but had no effect on sociability and preference for social novelty. Conditional deletion of CA2/CA3a Oxtr showed no changes in anxiety-like behavior assessed using the open-field, elevated plus maze and novelty-suppressed feeding tests. Application of a highly selective OXTR agonist [Thr4,Gly7]-OXT to hippocampal slices resulted in an acute and lasting potentiation of excitatory synaptic responses in CA2 pyramidal neurons that relied on N-methyl-d-aspartate receptor activation and calcium/calmodulin-dependent protein kinase II activity. In addition, Oxtr-/- mice displayed a defect in the induction of long-term potentiation, but not long-term depression, at the synapses between the entorhinal cortex and CA2 pyramidal neurons. Furthermore, Oxtr deletion led to a reduced complexity of basal dendritic arbors of CA2 pyramidal neurons, but caused no alteration in the density of apical dendritic spines. Considering that the methodologies we have used to delete Oxtr do not rule out targeting the neighboring CA3a region, these findings suggest that OXTR signaling in the CA2/CA3a is crucial for the persistence of long-term SRM.SIGNIFICANCE STATEMENT Oxytocin receptors (OXTRs) are abundantly expressed in hippocampal CA2 and CA3 regions, but there are little known about their physiological function. Taking advantage of the conditional Oxtr knock-out mice, the present study highlights the importance of OXTR signaling in the induction of long-term potentiation at the synapses between the entorhinal cortex and CA2 pyramidal neurons and the persistence of long-term social recognition memory. Thus, OXTRs in the CA2/CA3a may provide a new target for therapeutic approaches to the treatment of social cognition deficits, which are often observed in patients with neuropsychiatric disorders.

In Parkinson's disease patients and rodent models, dopaminergic neuron loss (DAN) results in severe motor disabilities. In contrast, general motility is preserved after early postnatal DAN loss in rodents. Here we used mice of both sexes to show that the preserved motility observed after early DAN loss depends on functional changes taking place in medium spiny neurons (MSN) of the dorsomedial striatum (DMS) that belong to the direct pathway (dMSN). Previous animal model studies showed that adult loss of dopaminergic input depresses dMSN response to cortical input, which likely contributes to Parkinson's disease motor impairments. However, the response of DMS-dMSN to their preferred medial PFC input is preserved after neonatal DAN loss as shown by in vivo studies. Moreover, their response to inputs from adjacent cortical areas is increased, resulting in reduced cortical inputs selectivity. Additional ex vivo studies show that membrane excitability increases in dMSN. Furthermore, chemogenetic inhibition of DMS-dMSN has a more marked inhibitory effect on general motility in lesioned mice than in their control littermates, indicating that expression of normal levels of locomotion and general motility depend on dMSN activity after early DAN loss. Contrastingly, DMS-dMSN inhibition did not ameliorate a characteristic phenotype of the DAN-lesioned animals in a marble burying task demanding higher behavioral control. Thus, increased dMSN excitability likely promoting changes in corticostriatal functional connectivity may contribute to the distinctive behavioral phenotype emerging after developmental DAN loss, with implications for our understanding of the age-dependent effects of forebrain dopamine depletion and neurodevelopment disorders.SIGNIFICANCE STATEMENT The loss of striatal dopamine in the adult brain leads to life-threatening motor impairments. However, general motility remains largely unaffected after its early postnatal loss. Here, we show that the high responsiveness to cortical input of striatal neurons belonging to the direct basal ganglia pathway, crucial for proper motor functioning, is preserved after early dopamine neuron loss, in parallel with an increase in these cells' membrane excitability. Chemogenetic inhibition studies show that the preserved motility depends on this direct pathway hyperexcitability/hyperconnectivity, while other phenotypes characteristic of this condition remained unaltered despite the dMSN inhibition. This insight has implications for our understanding of the mechanism underlying the behavioral impairments observed in neuropsychiatric conditions linked to early dopaminergic hypofunction.

Parental care is critical for successful reproduction in mammals. Recent work has implicated the hormone prolactin in regulating male parental behavior, similar to its established role in females. Male laboratory mice show a mating-induced suppression of infanticide (normally observed in virgins) and onset of paternal behavior 2 weeks after mating. Using this model, we sought to investigate how prolactin acts in the forebrain to regulate paternal behavior. First, using c-fos immunoreactivity in prolactin receptor (Prlr) Prlr-IRES-Cre-tdtomato reporter mouse sires, we show that the circuitry activated during paternal interactions contains prolactin-responsive neurons in multiple sites, including the medial preoptic nucleus, bed nucleus of the stria terminalis, and medial amygdala. Next, we deleted Prlr from three prominent cell types found in these regions: glutamatergic, GABAergic, and CaMKIIα. Prlr deletion from CaMKIIα, but not glutamatergic or GABAergic cells, had a profound effect on paternal behavior as none of these KO males completed the pup-retrieval task. Prolactin was increased during mating, but not in response to pups, suggesting that the mating-induced secretion of prolactin is important for establishing the switch from infanticidal to paternal behavior. Pharmacological blockade of prolactin secretion at mating, however, had no effect on paternal behavior. In contrast, suppressing prolactin secretion at the time of pup exposure resulted in failure to retrieve pups, with exogenous prolactin administration rescuing this behavior. Together, our data show that paternal behavior in sires is dependent on basal levels of circulating prolactin acting at the time of interaction with pups, mediated through Prlr on CaMKIIα-expressing neurons.SIGNIFICANCE STATEMENT Parental care is critical for offspring survival. Compared with maternal care, however, the neurobiology of paternal care is less well understood. Here we show that the hormone prolactin, which is most well known for its female-specific role in lactation, has a role in the male brain to promote paternal behavior. In the absence of prolactin signaling specifically during interactions with pups, father mice fail to show normal retrieval behavior of pups. These data demonstrate that prolactin has a similar action in both males and females to promote parental care.

Glutamate transporter 1 (GLT1) is the main astrocytic transporter that shapes glutamatergic transmission in the brain. However, whether this transporter modulates sleep-wake regulatory neurons is unknown. Using quantitative immunohistochemical analysis, we assessed perisomatic GLT1 apposition with sleep-wake neurons in the male rat following 6 h sleep deprivation (SD) or following 6 h undisturbed conditions when animals were mostly asleep (Rest). We found that SD decreased perisomatic GLT1 apposition with wake-promoting orexin neurons in the lateral hypothalamus compared with Rest. Reduced GLT1 apposition was associated with tonic presynaptic inhibition of excitatory transmission to these neurons due to the activation of Group III metabotropic glutamate receptors, an effect mimicked by a GLT1 inhibitor in the Rest condition. In contrast, SD resulted in increased GLT1 apposition with sleep-promoting melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus. Functionally, this decreased the postsynaptic response of MCH neurons to high-frequency synaptic activation without changing presynaptic glutamate release. The changes in GLT1 apposition with orexin and MCH neurons were reversed after 3 h of sleep opportunity following 6 h SD. These SD effects were specific to orexin and MCH neurons, as no change in GLT1 apposition was seen in basal forebrain cholinergic or parvalbumin-positive GABA neurons. Thus, within a single hypothalamic area, GLT1 differentially regulates excitatory transmission to wake- and sleep-promoting neurons depending on sleep history. These processes may constitute novel astrocyte-mediated homeostatic mechanisms controlling sleep-wake behavior.SIGNIFICANCE STATEMENT Sleep-wake cycles are regulated by the alternate activation of sleep- and wake-promoting neurons. Whether and how astrocytes can regulate this reciprocal neuronal activity are unclear. Here we report that, within the lateral hypothalamus, where functionally opposite wake-promoting orexin neurons and sleep-promoting melanin-concentrating hormone neurons codistribute, the glutamate transporter GLT1, mainly present on astrocytes, distinctly modulates excitatory transmission in a cell-type-specific manner and according to sleep history. Specifically, GLT1 is reduced around the somata of orexin neurons while increased around melanin-concentrating hormone neurons following sleep deprivation, resulting in different forms of synaptic plasticity. Thus, astrocytes can fine-tune the excitability of functionally discrete neurons via glutamate transport, which may represent novel regulatory mechanisms for sleep.

The medial septum implements cortical theta oscillations, a 5-12 Hz rhythm associated with locomotion and paradoxical sleep reflecting synchronization of neuronal assemblies such as place cell sequence coding. Highly rhythmic burst-firing parvalbumin-positive GABAergic medial septal neurons are strongly coupled to theta oscillations and target cortical GABAergic interneurons, contributing to coordination within one or several cortical regions. However, a large population of medial septal neurons of unidentified neurotransmitter phenotype and with unknown axonal target areas fire with a low degree of rhythmicity. We investigated whether low-rhythmic-firing neurons (LRNs) innervated similar or different cortical regions to high-rhythmic-firing neurons (HRNs) and assessed their temporal dynamics in awake male mice. The majority of LRNs were GABAergic and parvalbumin-immunonegative, some expressing calbindin; they innervated interneurons mostly in the dentate gyrus (DG) and CA3. Individual LRNs showed several distinct firing patterns during immobility and locomotion, forming a parallel inhibitory stream for the modulation of cortical interneurons. Despite their fluctuating firing rates, the preferred firing phase of LRNs during theta oscillations matched the highest firing probability phase of principal cells in the DG and CA3. In addition, as a population, LRNs were markedly suppressed during hippocampal sharp-wave ripples, had a low burst incidence, and several of them did not fire on all theta cycles. Therefore, CA3 receives GABAergic input from both HRNs and LRNs, but the DG receives mainly LRN input. We propose that distinct GABAergic LRNs contribute to changing the excitability of the DG and CA3 during memory discrimination via transient disinhibition of principal cells.SIGNIFICANCE STATEMENT For the encoding and recall of episodic memories, nerve cells in the cerebral cortex are activated in precisely timed sequences. Rhythmicity facilitates the coordination of neuronal activity and these rhythms are detected as oscillations of different frequencies such as 5-12 Hz theta oscillations. Degradation of these rhythms, such as through neurodegeneration, causes memory deficits. The medial septum, a part of the basal forebrain that innervates the hippocampal formation, contains high- and low-rhythmic-firing neurons (HRNs and LRNs, respectively), which may contribute differentially to cortical neuronal coordination. We discovered that GABAergic LRNs preferentially innervate the dentate gyrus and the CA3 area of the hippocampus, regions important for episodic memory. These neurons act in parallel with the HRNs mostly via transient inhibition of inhibitory neurons.