Loss of Adult 5-HT1A Autoreceptors Results in a Paradoxical Anxiogenic Response to Antidepressant Treatment.

Selective serotonin (5-HT) reuptake inhibitors (SSRIs) are first-line antidepressants but require several weeks to elicit their actions. Chronic SSRI treatment induces desensitization of 5-HT1A autoreceptors to enhance 5-HT neurotransmission. Mice (both sexes) with gene deletion of 5-HT1A autoreceptors in adult 5-HT neurons (1AcKO) were tested for response to SSRIs. Tamoxifen-induced recombination in adult 1AcKO mice specifically reduced 5-HT1A autoreceptor levels. The 1AcKO mice showed a loss of 5-HT1A autoreceptor-mediated hypothermia and electrophysiological responses, but no changes in anxiety- or depression-like behavior. Subchronic fluoxetine (FLX) treatment induced an unexpected anxiogenic effect in 1AcKO mice in the novelty suppressed feeding and elevated plus maze tests, as did escitalopram in the novelty suppressed feeding test. No effect was seen in wild-type (WT) mice. Subchronic FLX increased 5-HT metabolism in prefrontal cortex, hippocampus, and raphe of 1AcKO but not WT mice, suggesting hyperactivation of 5-HT release. To detect chronic cellular activation, FosB+ cells were quantified. FosB+ cells were reduced in entorhinal cortex and hippocampus (CA2/3) and increased in dorsal raphe 5-HT cells of 1AcKO mice, suggesting increased raphe activation. In WT but not 1AcKO mice, FLX reduced FosB+ cells in the median raphe, hippocampus, entorhinal cortex, and median septum, which receive rich 5-HT projections. Thus, in the absence of 5-HT1A autoreceptors, SSRIs induce a paradoxical anxiogenic response. This may involve imbalance in activation of dorsal and median raphe to regulate septohippocampal or fimbria-fornix pathways. These results suggest that markedly reduced 5-HT1A autoreceptors may provide a marker for aberrant response to SSRI treatment.SIGNIFICANCE STATEMENT Serotonin-selective reuptake inhibitors (SSRIs) are effective in treating anxiety and depression in humans and mouse models. However, in some cases, SSRIs can increase anxiety, but the mechanisms involved are unclear. Here we show that, rather than enhancing SSRI benefits, adulthood knockout (KO) of the 5-HT1A autoreceptor, a critical negative regulator of 5-HT activity, results in an SSRI-induced anxiety effect that appears to involve a hyperactivation of the 5-HT system in certain brain areas. Thus, subjects with very low levels of 5-HT1A autoreceptors, such as during childhood or adolescence, may be at risk for an SSRI-induced anxiety response.

Serotonin 1A receptors alter expression of movement representations.

Serotonin has a myriad of central functions involving mood, appetite, sleep, and memory and while its release within the spinal cord is particularly important for generating movement, the corresponding role on cortical movement representations (motor maps) is unknown. Using adult rats we determined that pharmacological depletion of serotonin (5-HT) via intracerebroventricular administration of 5,7 dihydroxytryptamine resulted in altered movements of the forelimb in a skilled reaching task as well as higher movement thresholds and smaller maps derived using high-resolution intracortical microstimulation (ICMS). We ruled out the possibility that reduced spinal cord excitability could account for the serotonin depletion-induced changes as we observed an enhanced Hoffman reflex (H-reflex), indicating a hyperexcitable spinal cord. Motor maps derived in 5-HT1A receptor knock-out mice also showed higher movement thresholds and smaller maps compared with wild-type controls. Direct cortical application of the 5-HT1A/7 agonist 8-OH-DPAT lowered movement thresholds in vivo and increased map size in 5-HT-depleted rats. In rats, electrical stimulation of the dorsal raphe lowered movement thresholds and this effect could be blocked by direct cortical application of the 5-HT1A antagonist WAY-100135, indicating that serotonin is primarily acting through the 5-HT1A receptor. Next we developed a novel in vitro ICMS preparation that allowed us to track layer V pyramidal cell excitability. Bath application of WAY-100135 raised the ICMS current intensity to induce action potential firing whereas the agonist 8-OH-DPAT had the opposite effect. Together our results demonstrate that serotonin, acting through 5-HT1A receptors, plays an excitatory role in forelimb motor map expression.

An immunohistochemical study of the serotonin 1A receptor in the hippocampus of subjects with Alzheimer's disease.

Alzheimer's disease (AD) is associated with neuronal degeneration, synaptic loss and deficits in multiple neurotransmitter systems. Alterations in the serotonin 1A (5-HT1A) receptor can contribute to impaired cognitive function in AD, and both in vitro binding and Positron emission tomography (PET) imaging studies have demonstrated that 5-HT1A receptors in the hippocampus/medial temporal cortex are affected early in AD. This neuropathological study examined the localization and immunoreaction intensity of 5-HT1A receptor protein in AD hippocampus with the goal to determine whether neuronal receptor levels are influenced by the severity of NFT severity defined by Braaks' pathological staging and to provide immunohistochemical confirmation of the binding assays and PET imaging studies. Subjects included AD patients and non-AD controls (NC) stratified into three Braaks' stages (Braak 0-II, NC; Braak III/IV and V/VI, AD). In the Braak 0-II group, 5-HT1A-immunoreactivity (ir) was prominent in the neuropil of the CA1 and subiculum, moderate in the dentate gyrus molecular layer (DGml), and low in the CA3 and CA4. No changes in 5-HT1A-ir were observed in the hippocampus of AD subjects in the Braak III/IV group. Hippocampal 5-HT1A-ir intensity was markedly decreased in the CA1 region in 6/11 (54.5%) subjects in the Braak V/VI group. Across all three groups combined, there was a statistically significant association between reduced 5HT1A-ir and neuronal loss in the CA1, but not in the CA3. The present data demonstrate that hippocampal 5-HT1A receptors are mainly preserved until the end-stage of NFT progression in AD. Thus, the utility of PET imaging using a 5-HT1A-specific radiolabeled probe as a marker of hippocampal neuronal loss may be limited to the CA1 field in advanced stage AD cases.

Alpha-Ca2+/calmodulin-dependent protein kinase II contributes to the developmental programming of anxiety in serotonin receptor 1A knock-out mice.

Mice lacking the serotonin receptor 1A [Htr1aknock-out (Htr1a(KO))] display increased innate and conditioned anxiety-related behavior. Expression of the receptor in the mouse forebrain during development is sufficient to restore normal anxiety-related behavior to knock-out mice, demonstrating a role for serotonin in the developmental programming of anxiety circuits. However, the precise developmental period as well as the signaling pathways and neural substrates involved in this phenomenon are unknown. Here, we show that pharmacological blockade of the receptor from postnatal day 13 (P13)-P34 is sufficient to reproduce the knock-out phenotype in adulthood, thus defining a role for serotonin in the maturation and refinement of anxiety circuits during a limited postnatal period. Furthermore, we identify increases in the phosphorylation of alpha-Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) at threonine 286 in the hippocampus of young Htr1a(KO) mice under anxiety-provoking conditions. Increases in alphaCaMKII phosphorylation were most pronounced in the CA1 region of the hippocampus and were localized to the extrasynaptic compartment, consistent with a tissue-specific effect of the receptor. No changes in alphaCaMKII phosphorylation were found in adult knock-out mice, suggesting a transient role of alphaCaMKII as a downstream target of the receptor. Finally, the anxiety phenotype was abolished when knock-out mice were crossed to mice in which alphaCaMKII phosphorylation was compromised by the heterozygous mutation of threonine 286 into alanine. These findings suggest that modulation of alphaCaMKII function by serotonin during a restricted postnatal period contributes to the developmental programming of anxiety-related behavior.

5-HT7 receptor deletion enhances REM sleep suppression induced by selective serotonin reuptake inhibitors, but not by direct stimulation of 5-HT1A receptor.

5-HT(7) receptors are involved in REM sleep and possibly in mood disorders. REM sleep suppression and antidepressant-like behavior is observed in 5-HT(7)(-/-) mice and in rats treated with 5-HT(7) receptor antagonists. We recently demonstrated that pharmacological blockade of 5-HT(7) receptors enhances REM sleep suppression and antidepressant-like behavior induced by citalopram in rodents. It has been hypothesized that the effect of citalopram on sleep is essentially mediated by the activation of 5-HT(1A) receptors. The present study investigates the impact of 5-HT(7) receptor gene deletion on the effect of various reuptake inhibitors on REM sleep and probes the role of 5-HT(1A) receptors in this response. Three SSRIs (citalopram, fluoxetine and paroxetine) but not the tricyclic antidepressant desipramine had a significantly stronger REM sleep suppressive effect in 5-HT(7)(-/-) mice compared to 5-HT(7)(+/+) mice. In contrast, REM sleep was similarly reduced in 5-HT(7)(+/+) mice and 5-HT(7)(-/-) mice after treatment with the 5-HT(1A) receptor agonist ipsapirone. Furthermore, both 5-HT(7)(+/+) and 5-HT(7)(-/-) mice displayed the same increase in REM sleep duration produced by the 5-HT(1A) receptor antagonist WAY-100635. These findings indicate that 5-HT(7) receptor deletion augments the effect of various SSRIs on REM sleep suppression and that this effect is distinct from those mediated via 5-HT(1A) receptors.

Enhanced activity of pyramidal neurons in the infralimbic cortex drives anxiety behavior.

We show that in an animal model of anxiety the overall excitation, particularly in the infralimbic region of the medial prefrontal cortex (IL), is increased and that the activity ratio between excitatory pyramidal neurons and inhibitory interneurons (AR PN/IN) is shifted towards excitation. The same change in AR PN/IN is evident for wildtype mice, which have been exposed to an anxiety stimulus. We hypothesize, that an elevated activity and the imbalance of excitation (PN) and inhibition (IN) within the neuronal microcircuitry of the prefrontal cortex is responsible for anxiety behaviour and employed optogenetic methods in freely moving mice to verify our findings. Consistent with our hypothesis elevation of pyramidal neuron activity in the infralimbic region of the prefrontal cortex significantly enhanced anxiety levels in several behavioural tasks by shifting the AR PN/IN to excitation, without affecting motor behaviour, thus revealing a novel mechanism by which anxiety is facilitated.

Altered photic and non-photic phase shifts in 5-HT(1A) receptor knockout mice.

The mammalian circadian clock located in the suprachiasmatic nucleus (SCN) is thought to be modulated by 5-HT. 5-HT is though to inhibit photic phase shifts by inhibiting the release of glutamate from retinal terminals, as well as by decreasing the responsiveness of retinorecipient cells in the SCN. Furthermore, there is also evidence that 5-HT may underlie, in part, non-photic phase shifts of the circadian system. Understanding the mechanism by which 5-HT accomplishes these goals is complicated by the wide variety of 5-HT receptors found in the SCN, the heterogeneous organization of both the circadian clock and the location of 5-HT receptors, and by a lack of sufficiently selective pharmacological agents for the 5-HT receptors of interest. Genetically modified animals engineered to lack a specific 5-HT receptor present an alternative avenue of investigation to understand how 5-HT regulates the circadian system. Here we examine behavioral and molecular responses to both photic and non-photic stimuli in mice lacking the 5-HT(1A) receptor. When compared with wild-type controls, these mice exhibit larger phase advances to a short late-night light pulse and larger delays to long 12 h light pulses that span the whole subjective night. Fos and mPer1 expression in the retinorecipient SCN is significantly attenuated following late-night light pulses in the 5-HT(1A) knockout animals. Finally, non-photic phase shifts to (+/-)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) are lost in the knockout animals, while attenuation of the phase shift to the long light pulse due to rebound activity following a wheel lock is unaffected. These findings suggest that the 5-HT(1A) receptor plays an inhibitory role in behavioral phase shifts, a facilitatory role in light-induced gene expression, a necessary role in phase shifts to 8-OH-DPAT, and is not necessary for activity-induced phase advances that oppose photic phase shifts to long light pulses.

Sucrose intake and fasting glucose levels in 5-HT(1A) and 5-HT(1B) receptor mutant mice.

Serotonin (5-HT)(1A) and 5-HT(1B) receptors have been implicated in the incidence and treatment of depression in part through the examination of animals lacking these receptors. Although these receptors have been repeatedly implicated in ingestive behavior there is little information about how 5-HT(1A) and 5-HT(1B) receptor mutant mice react to solutions of varying palatability. In the present experiment male and female 5-HT(1A) and 5-HT(1B) mutant and wild-type mice were presented with increasing concentrations of sucrose using a two-bottle choice procedure. In addition fasting blood glucose levels were assessed. Both male and female 5-HT(1B) mutant mice drank more sucrose than WT mice but also consumed more water. Female, but not male, 5-HT(1A) mutant mice similarly showed increased sucrose consumption, but did not demonstrate increased consumption of water. In addition, the pattern of increased sucrose consumption over genotype and sex was related to fasting blood glucose concentrations such that levels in male 5-HT(1B) mutant mice were reduced relative to wild-type and 5-HT(1A) mutant males, but similar to those of females. The findings in 5-HT(1B) mutant mice emphasize the role of the 5-HT(1B) receptor in regulating ingestive behavior, whereas female sex hormones and 5-HT(1A) receptors may interact to alter sucrose consumption in 5-HT(1A) mutant mice. In addition, these findings may have implications for the role of these receptors in the incidence and treatment of depression since the intake of sucrose has been used as an index of anhedonia in animal models of depression and antidepressant efficacy.

Behavior of Adult 5-HT1A Receptor Knockout Mice Exposed to Stress During Prenatal Development.

Chronic maternal stress during pregnancy can have long-term, detrimental consequences for the offspring. An understanding of the mechanisms responsible for mediating these effects is essential for devising therapeutic interventions. Here, we examined whether serotonin 1A receptor (5-HT1AR) mediates the effects of maternal stress on the behavioral outcomes of the offspring as adults. Heterozygous (HET) mouse dams were bred with HET males and were randomly assigned to stress or control groups. Pregnant dams in the stress group were exposed to a regime of chronic unpredictable stress from embryonic day 7 to 18. At two months of age, groups of male and female wildtype (WT), HET, and knockout (KO) offspring underwent a comprehensive behavioral test battery that included tests of social behavior, memory, aggression, anxiety, sensorimotor information processing, and exploratory and risk assessment behaviors. Independent of genotype, prenatal stress resulted in a change in locomotor activity and fear memory in male mice and a change in prepulse inhibition in female animals. 5-HT1AR KO affected anxiety in male mice, and fear memory and prepulse inhibition in female mice. 5-HT1AR genotype moderated the effects of maternal prenatal stress exposure on social behavior of male offspring and on activity levels of female offspring. Our findings indicate that 5-HT1A receptor availability can affect outcomes of the offspring resulting from maternal prenatal stress exposure, and that these effects are sex-specific.

Neurokinin 1 receptor antagonism requires norepinephrine to increase serotonin function.

The present studies examined the role of norepinephrine (NE) system in mediating the enhancement of 5-HT function produced by neurokinin (NK)1 receptor antagonism. Dorsal raphe 5-HT and locus coeruleus NE neurons were recorded in vivo in mice lacking NK1 receptors in wildtype mice pretreated with the NK1 antagonist RP67580 and its inactive enantiomer RP 68651. RP67580 and RP68651 were also tested on 5-HT neurons of mice lacking the 5-HT(1A) receptor. RP67580 increased the firing rate of 5-HT neurons in wildtype mice and in 5-HT(1A) null mutant mice to the same degree, thus indicating that the mechanism by which NK1 antagonists enhances 5-HT firing is independent of 5-HT(1A) receptors. NE neuronal burst activity was increased in NK1 null mutant and wildtype mice given RP67580, but not with RP68651. After NE depletion, RP67580 was ineffective in increasing 5-HT neuronal firing activity in NK1 wildtype mice, and the enhancement of 5-HT neuronal firing observed in NK1 null mutant mice was abolished. In conclusion, NE neurons are essential for the action of NK1 antagonists on 5-HT neurons. In addition, the desensitization of 5-HT(1A) autoreceptors produced by NK1 receptor antagonism is not critical for enhancing 5-HT neuronal firing.

State-dependent alterations in hippocampal oscillations in serotonin 1A receptor-deficient mice.

Mice lacking the serotonin 1A receptor (5-HT(1A)R) show increased levels of anxiety-related behavior across multiple tests and background strains. Tissue-specific rescue experiments, lesion studies, and neurophysiological findings all point toward the hippocampus as a potential mediator of the phenotype. Serotonin, acting through 5-HT(1A)Rs, can suppress hippocampal theta-frequency oscillations, suggesting that theta oscillations might be increased in the knock-outs. To test this hypothesis, local field potential recordings were obtained from the hippocampus of awake, behaving knock-outs and wild-type littermates. The magnitude of theta oscillations was increased in the knock-outs, specifically in the anxiety-provoking elevated plus maze and not in a familiar environment or during rapid eye movement sleep. Theta power correlated with the fraction of time spent in the open arms, an anxiety-related behavioral variable. These results suggest a possible role for the hippocampus, and theta oscillations in particular, in the expression of anxiety in 5-HT(1A)R-deficient mice.

Involvement of 5-HT1A receptors in prefrontal cortex in the modulation of dopaminergic activity: role in atypical antipsychotic action.

Atypical antipsychotics increase dopamine (DA) release in the medial prefrontal cortex (mPFC), an effect possibly involved in the superior effects of atypical versus classical antipsychotics on cognitive/negative symptoms. We examined the role of 5-HT1A receptors in the mPFC on the modulation of dopaminergic activity and the mesocortical DA release in vivo. The highly selective 5-HT1A agonist BAY x 3702 (BAY; 10-40 microg/kg, i.v.) increased the firing rate and burst firing of DA neurons in the ventral tegmental area (VTA) and DA release in the VTA and mPFC. The increase in DA release in both areas was potentiated by nomifensine coperfusion. The selective 5-HT1A antagonist WAY-100635 reversed the effects of BAY in both areas, and the changes in the VTA were prevented by frontocortical transection. The application of BAY in rat and mouse mPFC by reverse dialysis increased local extracellular DA at a low concentration (3 microM) and reduced it at a higher concentration (30 microM). Both effects disappeared in 5-HT1A knock-out mice. In the presence of bicuculline, BAY reduced DA release at all concentrations. The atypical antipsychotics clozapine, olanzapine, and ziprasidone (but not haloperidol) enhanced DA release in the mPFC of wild-type but not 5-HT1A knock-out mice after systemic and local (clozapine and olanzapine) administration in the mPFC. Likewise, bicuculline coperfusion prevented the elevation of DA release produced by local clozapine or olanzapine application. These results suggest that the activation of mPFC 5-HT1A receptors enhances the activity of VTA DA neurons and mesocortical DA release. This mechanism may be involved in the elevation of extracellular DA produced by atypical antipsychotics.

Blockade of 5-HT1A receptors by (+/-)-pindolol potentiates cortical 5-HT outflow, but not antidepressant-like activity of paroxetine: microdialysis and behavioral approaches in 5-HT1A receptor knockout mice.

Selective serotonin reuptake inhibitors like paroxetine (Prx) often requires 4-6 weeks to achieve clinical benefits in depressed patients. Pindolol shortens this delay and it has been suggested that this effect is mediated by somatodendritic 5-hydroxytryptamine (5-HT) 1A autoreceptors. However clinical data on the beneficial effects of pindolol are conflicting. To study the effects of (+/-)-pindolol-paroxetine administration, we used genetical and pharmacological approaches in 5-HT1A knockout mice (5-HT1A-/-). Two assays, in vivo intracerebral microdialysis in awake mice and the forced swimming test (FST), were used to assess the antidepressant-like effects of this drug combination. Basal levels of extracellular serotonin, 5-HT ([5-HT]ext) in the frontal cortex (FCX) and the dorsal raphe nucleus (DRN) did not differ between the two strains of mice, suggesting a lack of tonic control of 5-HT1A autoreceptors on nerve terminal 5-HT release. Prx (1 and 4 mg/kg) dose-dependently increased cortical [5-HT]ext in both genotypes, but the effects were greater in mutants. The selective 5-HT1A receptor antagonist, WAY-100635 (0.5 mg/kg), or (+/-)-pindolol (5 and 10 mg/kg) potentiated the effects of Prx (4 mg/kg) on cortical [5-HT]ext in 5-HT1A+/+, but not in 5-HT1A-/- mice. Similar responses were obtained following local intra-raphe perfusion by reverse microdialysis of either WAY-100635 or (+/-)-pindolol (100 microM each). In the FST, Prx administration dose-dependently decreased the immobility time in both strains of mice, but the response was much greater in 5HT1A-/- mice. In contrast, (+/-)-pindolol blocked Prx-induced decreases in the immobility time while WAY-100635 had no effect in both genotypes. These findings using 5-HT1A-/- mice confirm that (+/-)-pindolol behaves as an antagonist of 5-HT1A autoreceptor in mice, but its blockade of paroxetine-induced antidepressant-like effects in the FST may be due to its binding to other neurotransmitter receptors.

Variability in the benzodiazepine response of serotonin 5-HT1A receptor null mice displaying anxiety-like phenotype: evidence for genetic modifiers in the 5-HT-mediated regulation of GABA(A) receptors.

Benzodiazepines (BZs) acting as modulators of GABA(A) receptors (GABA(A)Rs) are an important group of drugs for the treatment of anxiety disorders. However, a large inter-individual variation in BZ sensitivity occurs in the human population with some anxiety disorder patients exhibiting diminished sensitivity to BZ and reduced density of GABA(A)Rs. The mechanism underlying BZ treatment resistance is not known, and it is not possible to predict whether an anxiety patient will respond to BZ. 5-hydroxytryptamine1A receptor (5-HT1AR) null mice (R-/-) on the Swiss-Webster (SW) background reproduce several features of BZ-resistant anxiety; they exhibit anxiety-related behaviors, do not respond to BZ, have reduced BZ binding, and have decreased expression of the major GABA(A)R subunits alpha1 and alpha2. Here, we show that R-/- mice on the C57Bl6 (B6) background also have anxiety phenotype, but they respond to BZ and have normal GABA(A)R subunit expression. This indicates that the 5-HT1AR-mediated regulation of GABA(A)R alpha subunit expression is subject to genetic modification. Hybrid SW/B6-R-/- mice also exhibit BZ-resistant anxiety, suggesting that SW mice carry a genetic modifier, which mediates the effect of the 5-HT1AR on the expression of GABA(A)Ralpha subunits. In addition, we show that this genetic interaction in SW mice operates early in postnatal life to influence the expression of GABA(A)R alpha subunits at the transcriptional level. These data indicate that BZ-resistant anxiety results from a developmental arrest of GABA(A)R expression in SW-R-/- mice, and a similar mechanism may be responsible for the BZ insensitivity of some anxiety patients.

Effects of lighting condition on circadian behavior in 5-HT1A receptor knockout mice.

Serotonin (5-HT) is an important regulator of the mammalian circadian system, and has been implicated in modulating entrained and free-running rhythms, as well as photic and non-photic phase shifting. In general, 5-HT appears to oppose the actions of light on the circadian system of nocturnal rodents. As well, 5-HT mediates, at least in part, some non-photic responses. The 5-HT1A, 1B and 7 receptors regulate these acute responses to zeitgebers. 5-HT also regulates some entrained and free-running properties of the circadian clock. The receptors that contribute to these phenomena have not been fully examined. Here, we use 5-HT1A receptor knockout (KO) mice to examine the response of the mouse circadian system to a variety of lighting conditions, including a normal light-dark cycle (LD), T-cycles, phase advanced LD cycles, constant darkness (DD), constant light (LL) and a 6 hour dark pulse starting at CT5. Relative to wildtype mice, the 5-HT1A receptor KO mice have lower levels of activity during the first 8h of the night/subjective night in LD and LL, later activity onsets on transient days during re-entrainment, shorter free-running periods in LL when housed with wheels, and smaller phase shifts to dark pulses. No differences were noted in activity levels during DD, alpha under any light condition, free-running period in DD, or phase angle of entrainment in LD. While the 5-HT1A receptor plays an important role in regulating photic and non-photic phase shifting, its contribution to entrained and free-running properties of the circadian clock is relatively minor.

Age-dependent effects of serotonin-1A receptor gene deletion in spatial learning abilities in mice.

The serotonin (5-hydroxytryptamine, 5-HT) receptor 1A is involved in many physiological functions, including the regulation of learning and memory by acting either as an autoreceptor located on 5-HT neurons (raphe nuclei) or as a heteroreceptor on non-5-HT neurons, mainly in the hippocampal formation. To investigate whether the effects of 5-HT via 5-HT1A receptors on learning are age-sensitive, we evaluated the performance of young-adult (3 months old) and aged (22 months old) 5-HT1A knockout (KO) mice and their homologous wild types (WT) in the hippocampal-dependent spatial reference memory version of the Morris water maze. We demonstrated that young-adult 5-HT1AKO mice exhibit an impairment in learning and retention of the spatial task, as compared to WT mice, without showing any sign of change in their sensori-motor and locomotor abilities or motivation. This genotype effect does not persist during aging. In fact, aged 5-HT1AKO mice seem to be slightly facilitated during the early stages of learning. These results are consistent with a possible prevalence of 5-HT1A raphe functions in learning and memory abilities of young-adult animals, since the effects of the mutation on mice performance (impairment) are opposite to those found after intra-raphe injection of 5-HT1A agonists (facilitation), and with data showing increased activity of 5-HT neurons in 5-HT1AKO mice. The reduced effect of the mutation in aged animals possibly reflects the lower efficacy of autoreceptors due to aging and/or a prevalence of hippocampal heteroreceptors.

Developmental effects of serotonin 1A autoreceptors on anxiety and social behavior.

The serotonin 1A receptor (5-HT1A) has a major role in modulating the effects of serotonin on mood and behavior. Previous studies have shown that knockout of 5-HT1A selectively in the raphe leads to higher levels of anxiety during adulthood. However, it remains unclear whether this phenotype is due to variation in receptor levels specifically during development or throughout life. To test the hypothesis that developmental sensitivity may underlie the effects of 5-HT1A on anxiety, we used an inducible transgenic system to selectively suppress 5-HT1A levels in serotonergic raphe neurons from post-natal days (P) 14 to P30, with a maximal reduction of 40% at P21 and return to regular levels by P30. This developmental decrease in receptor levels has long-lasting consequences, increasing anxiety and decreasing social investigation in adulthood. In addition, post-natal knockdown of autoreceptors leads to long-term increases in the excitability of serotonergic neurons, which may represent a mechanism underlying the effects of post-natal receptor variation on behavior later in life. Finally, we also examined the interplay between receptor variation and juvenile exposure to stress (applied from P14 to P21). Similar to receptor knockdown, juvenile exposure to stress led to increased anxiety phenotypes but did not exacerbate 5-HT1A knockdown-mediated anxiety levels. This work indicates that the effects of 5-HT1A autoreceptors on anxiety and social behaviors are developmentally mediated and suggests that natural variations in the expression of 5-HT1A may act during development to influence individual anxiety levels and contribute to susceptibility to anxiety disorders.

Postnatal maintenance of the 5-Ht1a-Pet1 autoregulatory loop by serotonin in the raphe nuclei of the brainstem.

BACKGROUND: Despite the importance of 5-HT1A as a major target for the action of several anxiolytics/antidepressant drugs, little is known about its regulation in central serotonin (5-hydroxytryptamine, 5-HT) neurons. RESULTS: We report that expression of 5-HT1A and the transcription factor Pet1 was impaired in the rostral raphe nuclei of mice lacking tryptophan hydroxylase 2 (Tph2) after birth. The downregulation of Pet1 was recapitulated in 5-Ht1a-/- mice. Using an explant culture system, we show that reduction of Pet1 and 5-HT1A was rescued in Tph2-/- brainstem by exogenous 5-HT. In contrast, 5-HT failed to rescue reduced expression of Pet1 in 5-Ht1a-/- brainstem explant culture. CONCLUSIONS: These results suggest a causal relationship between 5-HT1A and Pet1, and reveal a potential mechanism by which 5-HT1A-Pet1 autoregulatory loop is maintained by 5-HT in a spatiotemporal-specific manner during postnatal development. Our results are relevant to understanding the pathophysiology of certain psychiatric and developmental disorders.

Subtle alterations in breathing and heart rate control in the 5-HT1A receptor knockout mouse in early postnatal development.

We hypothesized that absence of the 5-HT(1A) receptor would negatively affect the development of cardiorespiratory control. In conscious wild type (WT) and 5-HT(1A) receptor knockout (KO) mice, we measured resting ventilation (Ve), oxygen consumption (Vo(2)), heart rate (HR), breathing and HR variability, and the hypercapnic ventilatory response (HCVR) at postnatal day 5 (P5), day 15 (P15), and day 25 (P25). In KO mice compared with WT, we found a 17% decrease in body weight at only P5 (P < 0.01) and no effect on Vo(2). Ve was significantly (P < 0.001) lower at P5 and P25, but there was no effect on the HCVR. Breathing variability (interbreath interval), measured by standard deviation, the root mean square of the standard deviation (RMSSD), and the product of the major (L) and minor axes (T) of the Poincaré first return plot, was 57% to 187% higher only at P5 (P < 0.001). HR was 6-10% slower at P5 (P < 0.001) but 7-9% faster at P25 (P < 0.001). This correlated with changes in the spectral analysis of HR variability; the low frequency to high frequency ratio was 47% lower at P5 but 68% greater at P25. The RMSSD and (L × T) of HR variability were ~2-fold greater at P5 only (P < 0.001; P < 0.05). We conclude that 5-HT(1A) KO mice have a critical period of potential vulnerability at P5 when pups hypoventilate and have a slower respiratory frequency and HR with enhanced variability of both, suggesting abnormal maturation of cardiorespiratory control.

Validation of the dimensionality emergence assay for the measurement of innate anxiety in laboratory mice.

The open field test is a common tool to measure innate anxiety in rodents. In the usual configuration of this test the animal is forced to explore the open arena and its behavior includes both anxiety and non-anxiety responses. However, the open arena is generally small and allows only limited expression of exploratory behavior. The recently developed dimensionality emergence assay in which an animal is housed in a home cage with free access to a large circular arena elicits graded exploration and promises to serve as a more ethological test of anxiety. Here we examined the predictive validity of this assay for anxiety-related measures in mice. First, we compared their behavior in the presence or absence of access to the home cage and found that mice with access to the home cage exhibited a gradual build-up in exploration of the arena while those without did not. Then we identified behavioral measures that responded to treatment with the anxiolytic drug diazepam. Diazepam altered several classical measures of innate anxiety, such as distance traveled and thigmotaxis, but also led to a dose-dependent acceleration of the build-up as reflected in a significantly reduced latency to attain several exploratory landmarks. Finally, we tested the utility of the dimensionality emergence assay in assessing alterations in innate anxiety reported in mice carrying a knockout allele for the serotonin 1A receptor (Htr1a). Our findings support the validity of the dimensionality emergence assay as a method to extract an expanded repertoire of behavioral measures for the assessment of anxiety in laboratory mice.