What do family caregivers of Alzheimer's disease patients desire in smart home technologies

Keywords: Smart homes, Dementia care, Alzheimer's disease, Family caregivers, Acceptability

Contrasted results of a wide survey. V. RIALLE (1), C. OLLIVET (2), C. GUIGUI (3), C. HERVÉ (4)

I. Introduction

This article presents a survey on the views and perceptions of innovative smart home technologies (SHT) by family caregivers (FCs) (including any voluntary caregiver) of patients with Alzheimer's disease or related disorders (AD), as well as what these FCs expect of these SHT.

Alzheimer's disease is not curable and strongly linked to ageing, and most AD patients live at home with the permanent help of a FC (mostly husband/wife or children). The illness features memory loss, and a slow disintegration of personality and physical control, with manifestation of aggressiveness, wandering, incontinence, disinhibition, binge-eating, hallucinations, delusions, and depression [START_REF] Nagaratnam | Behavioral and psychiatric manifestations in dementia patients in a community: caregiver burden and outcome[END_REF][START_REF] Larson | Cognitive impairment: dementia and Alzheimer's disease[END_REF][START_REF] Letenneur | Cerebral and functional aging: first results on prevalence and incidence of the Paquid cohort[END_REF]. As the illness develops, patients become totally dependant on their caregivers, who in turn may develop more or less severe physical exhaustion, negative thoughts, a depressive state, and are threatened by a higher mortality risk [START_REF] Takahashi | Depression and associated factors of informal caregivers versus professional caregivers of demented patients[END_REF][START_REF] Schulz | Caregiving as a risk factor for mortality: the Caregiver Health Effects Study[END_REF]. This stark reality is strongly contrasted by recent progress in smart homes technologies including communication and assistive technologies, and innovative telecare supports for elderly and cognitively impaired people [START_REF] Demiris | Home based E-health applications[END_REF][START_REF] Rialle | Health 'smart' home: Information technology for patients at home[END_REF][START_REF] Rialle | Smart Homes[END_REF][START_REF] Iliffe | Design and implementation of a computer decision support system for the diagnosis and management of dementia syndromes in primary care[END_REF][START_REF] Magnusson | A literature review study of Information and Communication Technology as a support of frail older people living at home and their family carers[END_REF]. Such progress is developing especially in the domain of wandering and fall detection, household robotics and domotics, user friendly videoconferencing communication for homecare and social connectedness, thus giving rise to innovative community telehealth network programs [START_REF] Rialle | Health 'smart' home: Information technology for patients at home[END_REF][START_REF] Magnusson | A literature review study of Information and Communication Technology as a support of frail older people living at home and their family carers[END_REF][START_REF] Bjorneby | Technology, ethics and dementia: A guidebook on how to apply technology in dementia care[END_REF][START_REF] Marshall | ASTRID: a social & technological response to meeting the needs of individuals with dementia and their carers[END_REF][START_REF] Czaja | Telecommunications technology as an aid to family caregivers of persons with dementia[END_REF][START_REF] Hanson | Case study: benefits of IT for older people and their carers[END_REF][START_REF]Enabling technologies for people with dementia[END_REF]. Today, despite markedly available new devices and the growing development of such programs, family caregivers remain physically and psychologically overstrained by the heavy care burden, which has a bearing on the quality of care they give. Distribution and uptaking of these new tools and resources is so poorly developed that personalized care plans and coping aids do not even mention them [START_REF] Marshall | Technology and technophobia[END_REF]. So, there is a need to better understand the hows and whys of the discrepancy between the available technology and the way AD FCs perceive it. Studies on needs, perceptions, and expectations of AD patients and their caregivers regarding assistive technologies have been studied so far through three main approaches: a) direct users' position through end user focus groups or in-depth face-to-face interviews; b) self-administered questionnaire; and c) ethnographic studies.

End user focus groups or in-depth face-to-face interviews are aimed at collecting the users' opinion through free expression, individually or in small groups. They are widely used by searchers or evaluators for the assessment of specific tools and services [START_REF] Demiris | Home based E-health applications[END_REF][START_REF] Anderson | Views of family carers and older people of information technology[END_REF][START_REF] Heller | Assessing Needs, Knowledge, Barriers And Use Of Technology Among Persons With Cognitive Disabilities[END_REF][START_REF] Haigh | The Independent LifeStyle AssistantTM[END_REF][START_REF] Hanson | Respite care for frail older people and their family carers: concept analysis and user focus group findings of a pan European Nursing research Project[END_REF]. They are usually costly and time consuming, and thus usable only for small scale assessment procedures.

The self-administered questionnaire approach is based on a set of questions, preferably very simple for the sake of understanding, sent to the target persons with a prepaid envelope for returning the filled questionnaire, or filled out over the telephone. This method is used in medium and large-scale studies for statistical purpose. It has been successfully applied to technology in the AD domain [START_REF] Haigh | The Independent LifeStyle AssistantTM[END_REF][START_REF] Demiris | A questionnaire for the assessment of patients' impressions of the risks and benefits of home telecare[END_REF].

The ethnographic study method consists in the in-depth understanding of people and context through direct observation of living situation by researchers. It was recently applied to the AD domain [START_REF] Haigh | The Independent LifeStyle AssistantTM[END_REF][START_REF] Morris | New Perspectives on Ubiquitous Computing from Ethnographic Study of Elders with Cognitive Decline[END_REF][START_REF] Salvador | Practical Considerations of Context for Context Based Systems: An Example from an Ethnographic Case Study of a Man Diagnosed with Early Onset Alzheimer's Disease[END_REF]. This method can only be applied to a small number of participants and a small-scale environment, thus raising two issues: the representativeness of the observed subjects, and the influence of the presence of the observer on observed people or situation. It is not rare to meet a mix of methodologies in extended studies [START_REF] Haigh | The Independent LifeStyle AssistantTM[END_REF].

One of the most thorough technological needs analyses was performed by K.Z. Haigh et al. [START_REF] Haigh | The Independent LifeStyle AssistantTM[END_REF]. They identified a list of nearly 300 technological appliances which would be of interest to elders, their caregivers (formal or informal), and other interested parties (e.g. insurance); opportunities of use were classified into general categories such as communications, activity monitoring, user monitoring, environment monitoring, reasoning, memory support, workload support, social support, event detection, and others. Then, by means of a Decision Matrix and Six Sigma (6σ) analyses, they determined the importance factor associated with each assistance need based on prevalence, contribution to institutionalization, impact on care giving resources, and limitations on elder functional ability. The ranked list of assistance needs based on an importance factor score (in parentheses) is the following: Medical monitoring (9.0), Medication management (7.5), Mobility (7.5), Caregiver burnout (7.5), Dementia (6.0), Eating (6.0), Toileting (6.0), Safety (6.0), Isolation (5.5), Transportation (5.5), Housekeeping (3.5), Money management Our study for a better understanding of FCs' perception of technology was named ALICE (for 'Alzheimer, Information, Communication and Ethics') and was performed on a large sample of French FCs of AD patients living in the greater Paris area. Medical gravity and social impact of AD in the French population has been thoroughly studied in recent years. For instance, the REAL.FR study was a prospective longitudinal study conducted by the French network on Alzheimer's disease [START_REF] Gillette-Guyonnet | The French network multicenter study. Predictive factors of hospitalization and management of Alzheimer's disease patients (REAL.FR)][END_REF], and the PAQUID survey was a prospective population-based cohort study on normal and pathological aging after 65 years which included around 1,500 elders aged 75 years and older [START_REF] Ramaroson | Prevalence of dementia and Alzheimer's disease among subjects aged 75 years or over: updated results of the PAQUID cohort[END_REF]. Furthermore Thomas et al. [START_REF] Thomas | Dementia patients caregivers quality of life: the PIXEL study[END_REF] conducted the PIXEL study, the goal of which was to demonstrate the parameters influencing French caregivers' quality of life, and their possible link with AD patients' quality of life.

The main features of the ALICE study sample are homogeneous with these earlier studies. For instance with regard to PIXEL: patients age range (PIXEL: 80.2 ± 6.8 [61-97]; ALICE: 78 ± 8.1 [54-98]); caregivers age range (PIXEL: 65.7 ± 12.8 [33-92]; ALICE: 64 ± 12.9 ); caregivers sex distribution (PIXEL: 63% women, 37% men; ALICE: 65.8% women, 34.2% men).

The following is a presentation of a specific part of the ALICE study: the cluster analysis of FCs' opinion based on answers to questions concerning 14 specific devices constituting a wide and representative range of SHT.

II. Materials and Methods

II.1 Population

The following three departments of the greater Paris area were selected for the study: Seine et Marne (Dpt # 77), Hauts de Seine (Dpt # 92), and Seine Saint Denis (Dpt # 93). They represent a large part of the Paris region including a number of satellite cities and rural areas. They were selected for their representativeness of the French social and cultural diversity: the first one is an urban and rural zone with scattered dwellings among crops; the second one has a population of well-off people and numerous head offices of big firms; the third one has a working population with a high percentage of immigrants. Each of these departments has its local Alzheimer's family association which provided the study with its own list of AD family member addresses for a total of 1,458 families.

II.2 Methods

Questionnaire

A self-administered questionnaire was written out, tested for reliability and validity, and sent by surface mail to 1,458 families of the selected population. It was intended to collect FCs' representations, wishes, and fears regarding a wide range of SHT, along with patients' abilities regarding commonly used devices. It was made-up of 50 questions divided in three main sections ( Besides usual and quite simple questions regarding the appreciation or rejection of the presented technologies, we tried to probe deeper on issues regarding the patient's intimacy and dignity, and the caregiver's questioning on the meaning of life, suffering, responsibility, and death. We tried to understand whether technology had any influence on these difficult questions. Most of the questions were composed of several elementary sub-questions. For instance question n° 18 corresponded to the 'mini-Zarit' score [START_REF] Da Rocha | Construction d'une échelle simplifiée pour la détection en médecine générale du fardeau de l'aidant d'une personne âgée dépendante[END_REF], which is composed of 7 yes/no sub-questions. The only person entitled to fill up the questionnaire was the family caregiver. Most of the questions relating to smart home technology were twofold: a first part was devoted to the caregiver's personal view, while a second part was devoted to the patient's view collected by the caregiver when possible. Because of data collection bias the latter were used only for a qualitative estimation of this view.

Subset of questions used for the cluster analysis

The subset of questions used for the cluster analysis is the "Viewpoint on specific technologies" (VST) part of the questionnaire (Tab. 1, section B-b). This subset was devoted to assess the FC's viewpoint on 14 specific SHT (Tab.2): fall sensor (3 questions: sewn in a belt or garment, stuck on the skin, inserted inside the body), oral call identification (1 q.), video surveillance (2q.: indoor, outdoor), tracking device (1q.), patient's assessment of functional abilities (2q.: displacement and transfer), activity of daily living (ADL) identification (1q.), cooking (2q.:

oral and written advices), robot care (1q.), videoconferencing (1q.). We purposely did not include 'domotic'

technologies such as remote door or window openers, since they are already widespread. In the same way, we did not try to be exhaustive with examples of innovative technologies since their number is currently growing considerably: the goal was to present an adequately varied and typical panel so as to reliably assess the caregiver's opinion.

Every question was of the multi-choice type and was structured as follows. First, it presented a short description of the technology followed by the question: "Would this technology be helpful to you?"), second, an agreement scale -

'not at all', 'little', 'moderately', 'very much' -as the set of possible answer choices.

II.3 Statistical methods used

Beside usual tests of independence for statistical relationship analyses (Chi Square, t-test, Anova), the study used the Multiple Correspondence Analysis (MCA) [START_REF] Benzecri | Correspondence analysis handbook[END_REF] to identify FC clusters. Each VST question corresponds to a VST variable of the MCA.

III. Results

III.1 Population

350 questionnaires were sent back by FCs, among which 270 (18.5% of the 1,458 questionnaires mailed to the families) met the completion criteria for the statistical analysis. Patients were 78.3 years old (± 8.1), range 54-98 years; 67.2% were women and 32.8% men. FCs were 64 years old (± 13), range 31-92 years. 65.8% were women and 34.2% men; a lot of them were very old; 13.9% were between 80 and 89 years of age (Fig. 1). 48% of FCs was the patient's husband or wife, 43.5% was a sibling, 2.6% was a relative, and 5.9% was other. The sex ratio of FCs (0.52) and patients (0.48) were comparable. 54.6% FCs had been the patient's caregiver for over 4 years, among whom 12.8% for over 8 years. 21% did not have any free time for themselves during the day and 11% had only one hour per day. 69.4% of the patients lived in a private home (individual house or apartment), 21.3% in a nursing home, a few in sheltered housing (2.2%), and 7.1% in specialized institutions or other. 52.3% of the patients had been diagnosed with Alzheimer's disease only two years after the onset of symptoms. 73.2% FCs considered their care recipient as very dependent (rate ≥ 5 on a 0-7 scale), and 38.8% as totally dependent (rate = 7 on a 0 to 7 scale).

Figure 1: Caregivers' (left) and patients' (right) age distribution III.2 Technology

Distributions of answers to VST questions (Tab. 2, and Fig. 2) can be characterized as follows: 11 questions among the 14 feature a U shaped distribution of answers, peaks of answers are on the extreme items "not at all" and "very much". For the three other questions (i.e., [START_REF] Salvador | Practical Considerations of Context for Context Based Systems: An Example from an Ethnographic Case Study of a Man Diagnosed with Early Onset Alzheimer's Disease[END_REF]34,35), the peaks are on "not at all" and "moderately", which flattens the U-shape. The means distribution is also U shaped.

The most appreciated technology, the tracking device (Q. 28), collected the greatest number of favorable responses (Tab. 2), with female caregivers appreciating it significantly more than male (p=0.0025, N=195) and younger caregivers more than older (p<0.001, N=196). The second most appreciated technology was the videoconferencing device for social connectedness (Q. 41) (Tab. 2), with no significant difference between male and female caregivers, or between younger and older ones. The least rejected device was the personal pocket videoconferencing device for mobile private remote surveillance of the patient (Q. 26) (Tab. 2), with also no significant difference between male and female caregivers, or between younger and older ones.

Concerning little appreciated technologies, a device for providing oral cooking advice (Q. 34) was given simultaneously the min. of 'very much' (6.4%) and the max. of 'not at all' (75.7%), and was closely followed by the fall sensor inserted inside the body under the skin ('not at all': 74.5%, 'very much': 10.8% MCA was used to determine the various FC classes suggested by the U shaped distribution of answers. The question was: were the respondents who selected 'not at all' and respectively 'very much' globally the same for the whole set of VST questions? If this was the case, they would make up a distinctive cluster of FCs. The first MCA factorial map (Fig. 3) proves that this is indeed the case: the "not at all" items and respectively 'very much' items are clearly grouped in homogenous clusters, and the two other clusters ('little' and 'moderately') can be easily localized even though they share a few borderline answerers.

Figure 3: Clustering of caregivers (0 ≡ not at all, 1 ≡ little, 2 ≡ moderately, 3 ≡ very much). The map shows the distribution of the answers to the 14 VST variables (N=90, questionnaires with a missing answer to at least one VST question were ignored, 23.47% of the total variance is "explained" by the map) Thus, two major opposite trends emerged from the collected data: the FCs who were mainly in favor of a substantial usage of technology for care aiding, and those who were rather or totally hostile to such a use. The tracking device was representative of such a bimodal distribution of opinions; the variance analysis resulted in a significant relation between the caregiver's age and the appreciation of the system (p<0.0001, N=214; mean of 'very much'= 60.7 y.o., of 'not at all'= 69.4 y.o.). This was also true for advanced devices such as company robots; sibling caregivers were more interested by these robots than the patient's husband or wife (p=0.005 ; N=243; mean of 'very much'= 57 y.o., of 'not at all'= 65 y.o.).

IV. Discussion

IV.1 Concerning the results

The results reinforce the conclusion reported by Poulson et al [START_REF] Poulson | USERfit: A Practical Handbook on User-centred Design for Assistive Technology[END_REF] according to whom there is no such things as an 'average user' in the area of assistive technologies. The fact that the two extreme items of VST questions ('not at all' and 'very much') were significantly more often selected than the medium ones ('little' and 'moderately') clearly suggests that family or voluntary caregivers are sensitive to technology. This closely matches the field reality for several technologies. For instance, the most appreciated one -tracking device (Q. 28) -is meant to answer to one of the most worrying issue of dementia care giving. According to Koester [START_REF] Koester | Lost Alzheimer's Disease Search Management: A law enforcement guide to managing the initial response and investigation of the missing Alzheimer's disease subject[END_REF], when a person with Alzheimer's disease or related disorders is lost for more than 12 hours, there is a 50% chance of finding him/her injured or dead.

Confronted with such a threat, caregivers must be constantly watchful, knowing that at any moment their cared-for is likely to wander or run away. Furthermore, the tracking device and service have long been awaited for by caregivers, despite ethical issues [START_REF] Mcshane | Tracking patients who wander: ethics and technology[END_REF].

The results also stress the importance of the videoconferencing device, both for social connectedness (second most appreciated, Q. 41) and for caring (least rejected, Q. 26) with no significant difference between male and female caregivers, or between younger and older ones, in both questions. The possibility to see, hear, and talk to the care recipient at a distance was considered as highly helpful.

IV.2 Concerning the methodology

Our study relies on an unusually large number of caregiver opinions obtained via a rather long self-administered questionnaire. Although a substantial amount of statistics was used to determine the quantitative features of the gathered data, the true intent of the study was not a statistical one. Statistics were used only to synthesize the voluminous collected information and to reveal the tendencies and remarkable relations from a statistical point of view, not to gain new academic knowledge on family caregivers. The results are those of a specific though widespread area -the greater Paris area -since it has a fairly diversified population, both culturally and economically. Moreover, these results represent only the opinions of the respondents and may not be generalizable to the entire group of FCs studied.

Due to the self-administered form of the enquiry and the age of caregivers, the first drawback of the methodology was the foreseeable difficulty for caregivers to properly understand the whole set of questions and to answer by the questionnaire themselves. The great number of questions and sub-questions increased this difficulty. The number of completely filled questionnaires (N=270, response rate = 18.5% of the 1,458 questionnaires mailed, as above mentioned) that were received in two months -without any letter of reminder -reinforce our conviction that the theme of the study was meaningful for caregivers (the total number of received answers was 351, among which 71 did not meet our filling criteria).

The second drawback of the self-administered questionnaire is the possible selection bias of the study. Indeed, it is conceivable that the people who were the most sensitive to the use of such technologies, either positively or negatively, were those most motivated to respond to the survey; these strong opinions then would appear as the result of the survey. These opinions might explain the U shaped distributions of responses. As a consequence, such a highly significant distribution shape might not be representative of the entire population. However, the hypothesis of such a bias is strongly weakened by the high rate of response obtained without any letter of reminder (18.5%), which is considered as more than expected (being usually subjectively expected around 10%). This response rate tends to prove that the inquiry was well accepted, and thus that every opinion tendency was correctly reflected in the received responses. 212 answers were received (the analysis of which would be too long for this article), which globally reflects a remarkable reflection, if not wisdom, on the part of family caregivers regarding technology. When expressed, their anger or resentment was most commonly directed against the poor consideration of the rest of the society for their burden of care and conditions of life.

V. Conclusion

Our study determined two major opposite trends in the opinions of family or voluntary caregivers concerning smart home technology, and provided further evidence of the contrasted status of technology in caregivers' perception:

rejection or mitigated appreciation of several technologies, and great confidence in the helpfulness of a few of these.

The study clearly shows that the more appreciated smart home technologies are those which increase the patient's safety while decreasing the caregiver's fear of wandering away or accident, and those which increase the caregiver's social connectedness and freedom to leave home at any time. Design of smart home technologies should take this into account to gain user acceptance. However further research will be needed to demonstrate the efficacy of technological resources in improving affect, coping, psychological well-being, and stress management, and to pass this data to family caregivers and health professionals. Such research should be carried out with an 'ethical intention' both to protect patients and caregivers against misuse [START_REF] Fisk | Telecare equipment in the home. Issues of intrusiveness and control[END_REF], and to maximize beneficial effects.

( 3 . 5 )

 35 , Shopping (3.5), Wandering (3.5), Usability (3.0), Equipment use (2.0), Hallucinations (2.0), Alcohol use (1.5), Pressure sores (1.0).

Figure 2 :

 2 Figure 2: Mean distribution of answers to the 14 VST questions "Would this technology be helpful to you?" along with three extreme answer distributions: personal videoconferencing (Q. 26), tracking device (Q. 28), and device providing oral cooking advice (Q. 34)

Table 1 )

 1 (the whole questionnaire is available on the ALICE website: http://www-timc.imag.fr/Vincent.Rialle/ALICE/Questionnaire_ALICE.pdf).

		Section	Topics	Questions #
	A)	General information Gender, demographics, lifestyle, and health status	1-18, 33, 46
	B)	Technology	(a) Current skills and practice	19-20, 31
			(b) Viewpoint on specific technologies	21-24, 26-28, 29a,
				29b, 30, 34-35, 39,
				41
			(c) Wishes, fears, meaning of life, and technology	25, 32, 36-38, 40,
				42-45
	C)	Economics	Economical effort and support	47-50

Table 1 :

 1 Composition of the questionnaire

Most of the questions were multi-choice. Only two of them were of the open-end type for free opinion expression.

Table 2 :

 2 ).

	not at all	little	Moderately	very much

Percentages of answers to the 14 VST questions "Would this technology be helpful to you?"

Acknowledgements

Our heartfelt thanks to the presidents and voluntary members of the three French departmental Alzheimer's disease associations (France Alzheimer 77, France Alzheimer 92, France Alzheimer 93; affiliated to the French national Association France Alzheimer) for the taskforce they devoted to the study. A special thanks also to Evelyne Janeau, of the Grenoble Joseph Fourier University, for her useful advice on data analysis tools.

Funding

The data collection and a part of the statistical analysis of the survey were funded by the Rotary Club of Le Raincy, France. The rest of the statistical and cluster analyses were funded by the Conseil Régional Rhône-Alpes.


Rémi Bos

Karina Sadlaoud

Pascale Boulenguez

Dorothée Buttigieg

Sophie Liabeuf

Cécile Brocard

Georg Haase

Hélène Bras

Laurent Vinay
email: laurent.vinay@univ-amu.fr.

Sylvie Liabeuf

Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

In healthy adults, activation of γ-aminobutyric acid (GABA) A and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl -] i ), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), E IPSP , in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT 2A receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT 2A R agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).

T he neuron-specific K + -Cl -cotransporter KCC2 (encoded by the solute carrier family 12 member 5, Slc12a5) extrudes Cl - and is responsible for the low [Cl -] i in mature neurons (1-3), a prerequisite for hyperpolarizing inhibition mediated by GABA A receptors (GABA A Rs) and glycine receptors (GlyRs). The expression or the function of KCC2 is reduced in several neurological disorders (2,4), and the resulting slight increase in [Cl -] i (depolarizing shift of the chloride equilibrium potential, E Cl ) dramatically compromises the inhibitory control of firing rate and excitatory inputs (5)(6)(7). Given the role of KCC2 in regulating the strength of inhibitory synaptic transmission, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance.

Spasticity is a disabling complication affecting individuals with spinal cord injury (SCI) and is characterized by a velocity-dependent increase in muscle tone resulting from hyperexcitable stretch reflexes, spasms, and hypersensitivity to normally innocuous sensory stimulations (8,9). Down-regulation of KCC2 after SCI in rats is implicated in the development of spasticity [START_REF] Boulenguez | Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury[END_REF] and chronic pain [START_REF] Cramer | The role of cation-dependent chloride transporters in neuropathic pain following spinal cord injury[END_REF][START_REF] Lu | Spinal cord injury-induced attenuation of GABAergic inhibition in spinal dorsal horn circuits is associated with downregulation of the chloride transporter KCC2 in rat[END_REF]. Notably, the expression of KCC2 in the motoneuron membrane is reduced, and, concomitantly, the density of cytoplasmic clusters is higher, suggesting that the surface stability of the transporter is reduced in these pathological conditions [START_REF] Boulenguez | Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury[END_REF].

Mounting evidence indicates that phosphorylation of KCC2 in the C-terminal intracellular domain dynamically regulates its activity and surface expression (1). In particular, phosphorylation by protein kinase (PK)C, enhances KCC2 activity and reduces endocytosis [START_REF] Lee | Direct protein kinase C-dependent phosphorylation regulates the cell surface stability and activity of the potassium chloride cotransporter KCC2[END_REF]. Interestingly, activation of 5-hydroxytryptamine type 2 receptors (5-HT 2 Rs) to serotonin stimulates PKC and strengthens the left-right alternation of motor bursts observed during locomotion [START_REF] Pearlstein | Serotonin refines the locomotor-related alternations in the in vitro neonatal rat spinal cord[END_REF][START_REF] Norreel | Reversible disorganization of the locomotor pattern after neonatal spinal cord transection in the rat[END_REF][START_REF] Jordan | Chapter 12-modulation of rhythmic movement: Control of coordination[END_REF], which rely on reciprocal inhibition [START_REF] Grillner | The motor infrastructure: From ion channels to neuronal networks[END_REF][START_REF] Kiehn | Locomotor circuits in the mammalian spinal cord[END_REF]. We hypothesized that 5-HT 2 R activity modulates KCC2 function and/ or expression. Our results indicate that the activation of the 5-HT 2A R subtype hyperpolarizes E IPSP via a PKC-dependent mechanism, increases KCC2 expression in the plasma membrane of motoneurons, and reduces SCI-induced spasticity.

Results

Negative Shift of E IPSP and Up-Regulation of KCC2. We first examined the effect of the 5-HT 2A/2B/2C R agonist (±)-2,5-dimethoxy-4iodoamphetamine hydrochloride (DOI) (10 μM; Table S1) on E IPSP in control neonatal rats [postnatal day (P)5-P7]. DOIhyperpolarized E IPSP within 10-20 min (Fig. 1 A andB). This effect was long-lasting (at least 2 h; Fig. 1B). E IPSP was significantly more hyperpolarized when motoneurons were recorded in the presence of DOI compared with control (8 mV; Fig. 1C,Left). There was a concomitant trend toward a depolarization of the resting membrane potential (V rest ) by DOI (+2 mV; P > 0.05). As a result, the amplitude of hyperpolarizing IPSPs recorded at V rest increased significantly (Fig. 1C, Right).

The next series of experiments was performed on animals that underwent a neonatal SCI. E IPSP was significantly more depolarized in those animals tested at P5-P7, compared with controls of the same age, as shown previously [START_REF] Jean-Xavier | Inhibitory postsynaptic potentials in lumbar motoneurons remain depolarizing after neonatal spinal cord transection in the rat[END_REF] (compare Fig. 1 C andD). Because of the increased sensitivity of neurons to 5-HT in those animals [START_REF] Norreel | Reversible disorganization of the locomotor pattern after neonatal spinal cord transection in the rat[END_REF], DOI was tested at a lower concentration (1-1.5 μM) than in controls. DOI induced an ∼8-mV hyperpolarization of E IPSP (Fig. 1D, Left). As a result, E IPSP shifted from above to below V rest (Fig. 1D, Right). Another set of animals was treated chronically with DOI from P4 to P6-P7 [0.15 mg/kg, i.p. [START_REF] Norreel | Reversible disorganization of the locomotor pattern after neonatal spinal cord transection in the rat[END_REF][START_REF] Sadlaoud | Differential plasticity of the GABAergic and glycinergic synaptic transmission to rat lumbar motoneurons after spinal cord injury[END_REF] twice a day]. E IPSP was more hyperpolarized in those DOI-treated animals than in untreated transected animals (Fig. 1E). Values were similar to those measured in control animals.

We performed subcellular fractionation of proteins from the lumbosacral spinal cord, followed by immunoblotting with a specific antibody against KCC2. The amount of KCC2 in the membrane fraction (KCC2Mb) was significantly increased after chronic DOI treatment, compared with NaCl-treated pups (Fig. 1F). There was a trend toward an increase in the amount of KCC2 in the cytosolic fraction. As a result, the ratio KCC2Mb/KCC2 cytoplasm was nonsignificantly increased. We then analyzed the expression of KCC2 by immunohistochemistry. All of the analyses were performed on a homogeneous population of retrogradely labeled lumbar motoneurons [triceps surae (TS) muscles (ankle extensors); Fig. 1G]. GlyRs are colocalized with the anchoring protein gephyrin and can, therefore, be used to label the plasma membrane; in addition, they are not affected by a neonatal spinal cord transection [START_REF] Sadlaoud | Differential plasticity of the GABAergic and glycinergic synaptic transmission to rat lumbar motoneurons after spinal cord injury[END_REF]. We, therefore, immunolabeled the α1 subunit of this receptor to visualize the plasma membrane of motoneurons in control (intact), untreated (SCI), and DOItreated cord-transected animals (SCI + DOI; Fig. 1G,Insets). After SCI, the intensity of KCC2 staining in the somatic membrane was reduced (Fig. 1H). DOI treatment increased KCC2 staining, so that no difference was observed compared with controls of the same age. We also quantified the KCC2 staining at the level of motoneuron dendrites that stretch out of the gray matter into the lateral and ventral funiculi (Fig. 1I). Similar to what was observed for the somatic membrane, DOI treatment restored the intensity of staining to values seen in controls (Fig. 1J). Altogether, these results indicate that the activation of 5-HT 2 R is able to restore chloride homeostasis (hyperpolarizing shift of E IPSP ) after neonatal SCI. An increased expression of KCC2 in the plasma membrane, at least in part, provides the molecular basis of this restoration.

Contribution of 5-HT 2A Rs. The effects of DOI on E IPSP were prevented in the presence of the 5-HT 2 R antagonist ketanserin (10 μM; n = 4; Fig. 1C,Center). Because DOI and ketanserin also bind to 5-HT 2B Rs and 5-HT 2C Rs (Table S1), to further identify the subtype of 5-HT 2 R that are involved, we used (4-bromo-3,6dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2), a high-affinity 5-HT 2A R agonist [START_REF] Mclean | 1-Aminomethylbenzocycloalkanes: Conformationally restricted hallucinogenic phenethylamine analogues as functionally selective 5-HT2A receptor agonists[END_REF][START_REF] Fox | The serotonin 5-HT(2A) receptor agonist TCB-2: A behavioral and neurophysiological analysis[END_REF] (Table S1). TCB-2 at low concentration (0.1 μM; n = 6) hyperpolarized E IPSP by ∼4 mV in control rats (Fig. 2 A and B, gray). Note that even values that were highly negative (-78 mV) were further hyperpolarized in the presence of TCB-2. The shift was larger (6.1 mV) at a higher concentration (10 μM; n = 4; Fig. 2B, black). TCB-2 had no effect on V rest and the other electrical properties investigated (Fig. S1). As a result, the driving force (E IPSP -V rest ) was significantly increased (Fig. 2B; -5.3 mV). We next tested this compound on motoneurons recorded from animals with neonatal SCI. The hyperpolarizing shift (-4.2 mV) induced by TCB-2 (0.1μM) was similar to that seen in control animals (Fig. 2C). On average, E IPSP switched from above to below V rest (Fig. 2C; P < 0.05; Wilcoxon paired test). The use of 5-HT 2A antagonists (ketanserin at 1 μM and MDL11,939 at 2 μM) and 5-HT 2B/2C agonists and antagonists confirmed the involvement of 5-HT 2A R in the negative shift of E IPSP and suggested that 5-HT 2B/2C may have opposite effects (Fig. S2). To identify the mechanisms underlying these effects, we used VU0240551 (25 μM), a highly specific KCC2 blocker [START_REF] Delpire | Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2[END_REF]. This compound depolarized E IPSP by >10 mV (Fig. 2D). KCC2 blockade occluded the effect of TCB-2 (Fig. 2D, Right), which is consistent with the requirement of KCC2 for the E IPSP shift induced by the activation of 5-HT 2A R. TCB-2 had no effect on the kinetics of IPSPs (Fig. S3), indicating that it modulates the strength of inhibition essentially through a modulation of KCC2 function.

We used immunolabeling to quantify the effect of TCB-2 on the expression of KCC2 at the plasma membrane of cultured motoneurons isolated from Hb9::eGFP transgenic mice (Fig. 2E,Left). Following activation of 5-HT 2A R by TCB-2 (0.1 μM; 30 min), the ratio of KCC2 expression between plasma membrane and cytosol, visualized by eGFP fluorescence (Fig. 2E, Insets), was strongly increased (Fig. 2E, Right).

PKC-Dependent Pathway Is Involved. We next investigated the second messenger pathway activated by TCB-2/DOI. 5-HT 2A Rs stimulate phospholipase C, leading to activation of PKC. Consistent with an involvement of PKC, the effects of both TCB-2 (Fig. 2F, black) and DOI (Fig. 2F, gray) were prevented by preincubation of PKC inhibitor chelerythrine (20 μM). In addition, activation of PKC with phorbol 12,13-dibutyrate (PDBu) (1 μM) induced an ∼9-mV negative shift of E IPSP (Fig. 2F), which switched on average from above (5.5 ± 3.7 mV) to below (-3.3 ± 2.1 mV) V rest . PKCs are divided into subfamilies, based on their second messenger requirements. The indolocarbazole Gö6976 enables discrimination between Ca 2+ -dependent and -independent isoforms of PKC. Nanomolar concentrations of this compound inhibit the Ca 2+ -dependent isozymes, whereas even micromolar concentrations have no effect on the Ca 2+ -independent PKC subtypes [START_REF] Martiny-Baron | Selective inhibition of protein kinase C isozymes by the indolocarbazole Gö 6976[END_REF]. Gö6976 (2 μM) induced a small (∼1.5 mV) but significant hyperpolarization of E IPSP (Fig. 2F). Importantly, a further ∼4-mV negative shift was observed after adding TCB-2 (Fig. 2F). The potent and selective activator of the Ca 2+ -independent PKCe, FR236924, induced an ∼2-mV negative shift of E IPSP (Fig. 2F). Altogether, these results demonstrate that the activation of 5-HT 2A R increases KCC2 function, at least in part, through a Ca 2+ -independent PKC. Activation of 5-HT 2A R Increases the Strength of Inhibition. Supramaximal stimulation of a lumbar dorsal root elicits a response in the homonymous ipsilateral ventral root in vitro (Fig. 3A). The earliest component representing the monosynaptic excitation of motoneurons [START_REF] Fulton | Electrophysiological properties of neonatal rat motoneurones studied in vitro[END_REF][START_REF] Pinco | In vitro studies of prolonged synaptic depression in the neonatal rat spinal cord[END_REF] was not affected by TCB-2 (0.1-0.15 μM; Fig. 3 A andB). The amplitude of the monosynaptic response decreases when the dorsal root is stimulated repeatedly (Fig. 3A), an effect that gets stronger as the frequency of stimulation increases [rate-dependent depression (RDD); Fig. 3C]. Because KCC2 plays a key role in the depression of the response (Fig. S4), we hypothesized that hyperpolarizing E IPSP by TCB-2 would affect the RDD. As expected, TCB-2 reduced the amplitude of the monosynaptic response elicited by repetitive stimulation (Fig. 3 A, blue trace, and Fig. 3C, dotted lines). As a result, the RDD was significantly increased (Fig. 3D). This effect was prevented by VU0240551 (Fig. S5).

We next considered the polysynaptic response, which results from a mixture of excitation and inhibition in motoneurons [START_REF] Wallis | FAST and SLOW ipsilateral and contralateral spinal reflexes in the neonate rat are modulated by 5-HT[END_REF][START_REF] Jiang | An in vitro functionally mature mouse spinal cord preparation for the study of spinal motor networks[END_REF]. The polysynaptic response is potentiated by VU0240551 within 15-20 min, as a result of decreased strength of inhibition (Fig. S6). This effect was reversible. TCB-2 reduced the polysynaptic response within 15-20 min, and this effect lasted at least 1 h (Fig. 3E), an effect that was occluded by prior application of picrotoxin and strychnine (to block GABA A Rs and GlyRs, respectively) or VU0240551 (Fig. S6). Therefore, TCB-2 increases the strength of postsynaptic inhibition in the intact spinal cord in vitro likely through a modulation of chloride homeostasis. In addition, such strengthening of inhibition by TCB-2 enabled to restore the alternating locomotor pattern that is disorganized after neonatal SCI (15) (Fig. S7).

Reduction of Spasticity After SCI. The H (or Hoffmann) reflex is commonly used to assess the excitability of the monosynaptic reflex loop in individuals suffering from spasticity. The H wave (Fig. 4A) resulting from the monosynaptic activation of motoneurons by type Ia afferents undergoes an RDD when using frequencies of stimulation higher than 0.1 Hz (29) (Fig. 4 A-C). Reduction of the RDD is a reliable correlate of spasticity after SCI [START_REF] Boulenguez | Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury[END_REF][START_REF] Thompson | Altered patterns of reflex excitability subsequent to contusion injury of the rat spinal cord[END_REF][START_REF] Grey | Post-activation depression of soleus stretch reflexes in healthy and spastic humans[END_REF]. The down-regulation of KCC2 accounts, at least in part, for this reduction [START_REF] Boulenguez | Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury[END_REF]. We investigated the effects of TCB-2 (0.3 mg/kg, i.p.) on the RDD in paraplegic spastic adult rats (14-21 d post-SCI). The RDD remained quite stable after i.p. injection of the vehicle (NaCl; Fig. 4 B andC). Injection of TCB-2 did not affect the thresholds, maximal amplitudes, or stimulus intensities at which the maximal amplitude is obtained for both M and H waves at 0.1 Hz (Table S2). However, an effect was clearly present when frequencies of stimulation higher than 0.5 Hz were used (Fig. 4 B andC). Indeed, the amplitude of the H wave was smaller than it was before injection, with a maximal and highly significant effect 7-25 min after TCB-2 injection (Fig. 4 B andC).

Discussion

This study demonstrates that the activation of a receptor to a neurotransmitter restores endogenous inhibition in pathological conditions by up-regulating KCC2 function. Activation of 5-HT 2A R by TCB-2 in vitro leads to a hyperpolarizing shift of E IPSP and an increased cell surface expression of KCC2 within minutes. Blocking KCC2 occluded this shift, indicating that the effect of TCB-2 is likely mediated by posttranslational modification of KCC2, as opposed to changes in gene transcription or protein synthesis. Chronic activation of 5-HT 2A R also induced a hyperpolarizing shift of E IPSP and a concomitant increased expression of KCC2. Changes in gene transcription or protein synthesis may occur in this situation, as suggested by the trend toward an increase in cytoplasmic KCC2.

Modulation of chloride homeostasis by 5-HT has been previously reported in the spinal cord [in zebrafish [START_REF] Brustein | Serotoninergic modulation of chloride homeostasis during maturation of the locomotor network in zebrafish[END_REF]] and several other systems (e.g., refs. 32 and 33). Here, we showed that the effect of 5-HT 2A R activation on chloride homeostasis was critically dependent on PKC. In agreement with these findings, the activity of a number of protein kinases, including PKC and phosphatases, has been reported to influence KCC2 function. It was further demonstrated that KCC2 is directly phosphorylated by PKC activity and that the major site of phosphorylation within the C-terminal intracellular domain of this protein is serine 940 (Ser940). PKCdependent phosphorylation of Ser940 increases KCC2 cell surface stability and activity by decreasing endocytosis from the plasma membrane [START_REF] Lee | Direct protein kinase C-dependent phosphorylation regulates the cell surface stability and activity of the potassium chloride cotransporter KCC2[END_REF]. In HEK-293 cells, the entire cell surface population of KCC2 is internalized over a time course of 10 min, a process that is dramatically slowed by activation of PKC [START_REF] Lee | Direct protein kinase C-dependent phosphorylation regulates the cell surface stability and activity of the potassium chloride cotransporter KCC2[END_REF]. Similarly, in cultured hippocampal neurons, PKC activation increases the cell surface expression of KCC2.

Activation of 5-HT 2A R still induced a hyperpolarizing shift of E IPSP after blocking Ca 2+ -dependent PKC by Gö6976. In addition, this compound induced a small hyperpolarizing shift of E IPSP . These results suggest opposite effects of Ca 2+ -dependent and Ca 2+ -independentxPKC on KCC2 function. In support of this hypothesis, repetitive postsynaptic spiking induces a positive shift in E Cl through a Ca 2+ -dependent down-regulation of KCC2 [START_REF] Fiumelli | Modulation of GABAergic transmission by activity via postsynaptic Ca2+-dependent regulation of KCC2 function[END_REF][START_REF] Woodin | Coincident pre-and postsynaptic activity modifies GABAergic synapses by postsynaptic changes in Cl-transporter activity[END_REF][START_REF] Fiumelli | Role of activity-dependent regulation of neuronal chloride homeostasis in development[END_REF].

Inhibition of Ca 2+ -dependent PKC abolishes the spiking-induced shift in E Cl [START_REF] Fiumelli | Modulation of GABAergic transmission by activity via postsynaptic Ca2+-dependent regulation of KCC2 function[END_REF]. In agreement with a negative effect of intracellular Ca 2+ on KCC2 function, NMDA receptor activation leads to Ca 2+ influx that ultimately causes protein phosphatase 1-mediated dephosphorylation of KCC2 residue Ser940 and depolarizing shift of E Cl [START_REF] Lee | NMDA receptor activity downregulates KCC2 resulting in depolarizing GABAA receptor-mediated currents[END_REF]. We showed that blocking 5-HT 2B Rs and 5-HT 2C Rs hyperpolarized E IPSP . Conversely, activating these receptors induced a depolarizing shift of E IPSP . Although we did not determine whether these effects are mediated by one or both of these receptors, these results suggest that 5-HT 2A Rs and 5-HT 2B/2C Rs have opposite effects on KCC2 function. Although the pharmacological characteristics of 5-HT 2A Rs and 5-HT 2C Rs are quite similar, owing, in part, to the high level of amino acid sequence homology between them (38), significant differences have been reported between the signaling cascades regulated by these receptors [START_REF] Berg | Signal transduction differences between 5-hydroxytryptamine type 2A and type 2C receptor systems[END_REF][START_REF] Berg | Differences in rapid desensitization of 5-hydroxytryptamine2A and 5-hydroxytryptamine2C receptor-mediated phospholipase C activation[END_REF]. Consistent with these observations, 5-HT 2A Rs and 5-HT 2C Rs exert opposing effects on both locomotor activity in mice [START_REF] Halberstadt | 5-HT(2A) and 5-HT(2C) receptors exert opposing effects on locomotor activity in mice[END_REF] and the long-lasting amplification of spinal reflexes in rats [START_REF] Machacek | Serotonin 5-HT(2) receptor activation induces a long-lasting amplification of spinal reflex actions in the rat[END_REF]. Interestingly, 5-HT 2B Rs and 5-HT 2C Rs become constitutively active (spontaneously active without 5-HT) after SCI [START_REF] Murray | Recovery of motoneuron and locomotor function after spinal cord injury depends on constitutive activity in 5-HT2C receptors[END_REF][START_REF] Murray | Motoneuron excitability and muscle spasms are regulated by 5-HT2B and 5-HT2C receptor activity[END_REF]. This constitutive activity may be partly responsible for the depolarizing shift of E Cl after SCI [START_REF] Boulenguez | Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury[END_REF]. The modulatory effects of 5-HT 2B/2C Rs on chloride homeostasis require furtherinvestigation.

The activation of 5-HT 2A R reduced spasticity after SCI, as revealed by the increased RDD of the H reflex. This effect was very likely due to the restoration of endogenous inhibition, because slight changes in E Cl were shown to have an important impact on the strength of inhibition (5-7, 10). 5-HT 2A Rs are strongly expressed in the ventral horn [START_REF] Pearlstein | Contribution of 5-HT to locomotionthe paradox of Pet-1(-/-) mice[END_REF] and are mainly localized in the plasma membrane of neurons, covering a large surface of cell bodies and dendrites [START_REF] Doly | The 5-HT2A receptor is widely distributed in the rat spinal cord and mainly localized at the plasma membrane of postsynaptic neurons[END_REF]. These receptors are dramatically up-regulated in the motoneuron somata and dendrites following SCI [START_REF] Kong | Robust upregulation of serotonin 2A receptors after chronic spinal transection of rats: An immunohistochemical study[END_REF], an effect that starts as early as 1 d after transection [START_REF] Kong | The time course of serotonin 2A receptor expression after spinal transection of rats: An immunohistochemical study[END_REF]. The 5-HT 2A R displays ∼10-fold lower constitutive activity than the 5-HT 2C R [START_REF] Aloyo | Current status of inverse agonism at serotonin2A (5-HT2A) and 5-HT2C receptors[END_REF].

In the intact spinal cord, the neuromodulators released by descending pathways allow the motoneurons to exhibit sustained depolarizations (plateau potentials), which are attributable to slowly activating voltage-dependent persistent inward currents (PICs). Shortly after the injury, the excitability of the spinal cord decreases because this excitatory influence of the brainstem on motoneurons is lost. However, motoneurons slowly recover the capacity to generate PICs, which are, in large part, responsible for longlasting reflexes and spasms [START_REF] Bennett | Spastic long-lasting reflexes in the awake rat after sacral spinal cord injury[END_REF][START_REF] Gorassini | Role of motoneurons in the generation of muscle spasms after spinal cord injury[END_REF]. Both 5-HT 2B Rs and 5-HT 2C Rs, but not 5-HT 2A Rs, are involved in this up-regulation of PICs [START_REF] Murray | Motoneuron excitability and muscle spasms are regulated by 5-HT2B and 5-HT2C receptor activity[END_REF]. To conclude, by restoring endogenous inhibition through an upregulation of KCC2 function, without depolarizing motoneurons and activating PICs, activation of 5-HT 2A Rs may represent an innovative therapeutic strategy to reduce spasticity after SCI. However, these receptors have been implicated in several psychiatric disorders (e.g., 51), and their effects on pain processing are controversial [START_REF] Millan | Descending control of pain[END_REF], highlighting the need to further investigate the potential systemic effects of specific 5-HT 2A R agonists, such as TCB-2.

Materials and Methods

In Vitro Electrophysiological Recordings. After hypothermic anesthesia, we dissected out the spinal cords (sacral segments up to T8-T9) and L3-L5 dorsal and ventral roots of newborn rats. Dissections and electrophysiological procedures were performed under continuous perfusion with an oxygenated artificial CSF.

In Vivo Electrophysiological Recordings. We measured the H reflex in adult rats under ketamine anesthesia (100 mg/kg, i.p.) using a pair of stainless steel needle electrodes transcutaneously inserted into the vicinity of the tibial nerve for stimulation. We placed the recording electrode into the flexor digitorum muscle beneath the ankle and the reference electrode s.c. into the foot.

Western Blots. The amount of KCC2 in the lumbar enlargement of the spinal cord was determined by Western blots as described previously [START_REF] Boulenguez | Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury[END_REF]. The procedures used for separating membrane and cytoplasmic fractions are detailed in SI Materials and Methods.

Immunohistochemistry. Fast blue (FB) [3 μL; 0.5% in 0.9% NaCl; F-5756 (Sigma)] was injected bilaterally in the TS muscles (ankle extensors) anesthetized animals at P5 for retrograde identification. Animals were killed 2 d later. After fixation, spinal cords were cut transversally (20-μm-thick sections). Immunohistochemistry was performed by using a mixture of affinity-purified rabbit

SI Materials and Methods

Animals. Wistar rats were purchased from Janvier. Hb9::eGFP and Slc12a5 +/-transgenic mice were kindly provided by T. Jessel (Columbia University, New York, NY) and E. Delpire (Vanderbilt University Medical Center, Nashville, TN), respectively. Animals were housed in a temperature-controlled animal care facility with a 12 h light-dark cycle. Generation and genotyping of the transgenic mice used have been described previously (1). We made all efforts to minimize animal suffering and the number of animals used. We performed experiments in accordance with French regulations (Ministry of Food, Agriculture and Fisheries, Division of Health and Protection of Animals). The local Direction of Veterinary Services and Ethical Committee (Marseille, Provence) delivered the appropriate licenses and approved the protocols, respectively.

Surgery. We anesthetized female rats (220-280 g) with a mixture of ketamine (60 mg/kg, i.p.; Imalgene; Merial) and medetomidine (0.25 mg/kg, i.p.; Domitor; Pfizer) and neonates by hypothermia. Amoxicillin, a long-lasting antibiotic, was administered immediately after anesthesia (150 mg/kg, s.c.; Clamoxyl LA; Pfizer). After laminectomy, we transected the spinal cord at the thoracic level (T8-T9). We treated sham-operated animals in the same way except for the spinal cord transection. Postsurgical care and treatments are described below. The surgery for transecting the spinal cord in neonates was similar to that described previously (2)(3)(4).

Postsurgical Treatments and Care of the Animals. After SCI in adult rats, we injected 5 mL of NaCl s.c., kept the rats warm until awakening, and placed some food with glucose in the cage. We administered buprenorphine to the rats every 8 h for 24 h (0.05 mg/ kg, s.c.; Temgesic; Reckitt Benckiser Healthcare) and diluted DL- lysine acetylsalicylate in their drinking water for 3 d (200 mg in 150 mL of drinking water; Aspégic nourissons; Sanofi Aventis). Twice a day, we manually emptied their bladders until recovery of the urinary function, checked their temperature and hydration, and observed any clinical sign of pain or infection. No urinary infection occurred. We killed rats presenting a more than 20% decrease in body weight, a temperature under 35 °C, or signs of autophagia with a lethal dose of pentobarbital.

Chronic Pharmacological Treatment. We activated 5-HT 2 Rs in animals with a SCI at birth by daily i.p. administration of the 5-HT 2 R agonist DOI, from P4 to P6-P7 [0.15 mg/kg DOI diluted in 50 μL of NaCl (2,4)]. Sham-treated animals were injected with 50 μL of NaCl only.

Western Blot. We euthanized animals by decapitation and quickly dissected out and froze T8-sacral parts of the spinal cords. For each sample, two spinal cords were pooled. For cytoplasmic and membrane fractions, we homogenized samples in cold lysis buffer B (described below) without detergent and centrifuged them first at 7,000 × g for 5 min and then at 18,000 × g for 70 min at 4 °C. We collected pellets (membrane-enriched fractions) in lysis buffer A and supernatants (cytoplasmic fractions). We determined supernatant protein concentrations using a detergent-compatible protein assay (Bio-Rad). We then immunoprecipitated KCC2 (described below), separated samples in 6% (vol/vol) SDS/ PAGE from 40% Acryl/Bisacrylamide (29/1) commercial solution, and transferred them to a PVDF membrane. After blockade in Tris-buffered saline-5% nonfat dry milk, we exposed membranes overnight at 4 °C to a polyclonal rabbit KCC2-specific antibody diluted at 1:700 (Millipore) in the blocking solution. The specificity of the KCC2 antibody to KCC2 is confirmed by the lower intensity of the signal in Slc12a5 +/-and Slc12a5 -/-mice (5).

We used an ImmunoPure goat HRP-conjugated rabbit-specific antibody (1:120,000 in blocking solution; 1 h at 22 °C; Pierce Biotech) for chemiluminescent detection (Millipore). Signal intensities were measured with the image analysis software Quantity-One (Bio-Rad). We incubated the same amount of protein with an affinity-purified rabbit KCC2-specific polyclonal antibody (Millipore) for 4 h at 4 °C and precipitated the protein-antibody complexes with PureProteome protein G magnetic beads (Millipore). After several washes in lysis buffer, beads were resuspended in SDS sample buffer.

Immunohistochemistry and Quantification of KCC2 Labeling. For immunohistochemistry, we anesthetized the animals at P7 by hypothermia, and killed them by decapitation (n = 3 in each group). We rapidly excised the lumbar spinal cord, immersed it for 1 h in 20% sucrose, and embedded it in capsules filled with Tissutech (Sakura) quickly frozen by immersion in ethanol 100% kept at -80 °C. We cut transverse spinal cord sections (20 μm thick) with a cryostat (Microm) and mounted them onto gelatinized slides. We immersed section in 2% paraformaldehyde in 0.15 M PBS (pH 7.4) for 30 min and rinsed in PBS. We incubated sections overnight at 4 °C in a mixture of affinity-purified rabbit KCC2-specific polyclonal antibody (1:200; Millipore) and GlyR monoclonal antibody (mAb2b; 1:100; Synaptic Systems). We then revealed the labeling with a mixture of donkey Cy3-conjugated rabbit-specific antibody (1:500; Jackson Immunoresearch) and donkey AlexaFluor 488conjugated mouse-specific antibody (1:800, Molecular Probes; 1 h at 22 °C) and mounted coverslips with a gelatinous aqueous medium. We analyzed the patterns of immunolabeling by means of a laser scanning confocal microscope (SM 510 META; Zeiss) at low (20×) or high magnification ([63× 1.4 (NA) oil immersion objective; Plan Apochromat].

Triple-fluorescent labelings were captured using frame-channel mode to avoid any cross-talk between the channels. Each optical section resulted from two scanning averages. Excitation of the fluorochromes was performed with a diode laser set at 405 nm to detect FB, an argon ion laser set at 488 nm, and a helium/neon laser set at 575 nm. At high magnification, we only scanned FBretrogradely labeled TS motoneurons with visible nuclei, and for each soma, we digitized stacks of 1-μm-thick optical sections. Because the developmental up-regulation of GlyR is not affected by neonatal transaction (2), we used the GlyR immunolabeling to delimit the somatic membrane of the motoneurons in sham-operated, transected, and DOI-treated transected animals, respectively. We overlaid the internal and external borders and digitized intensity measurements of KCC2 labeling using the Fluoview software (Version 5; Olympus). To quantify KCC2 labeling in the dendrites, we framed a zone of the white matter underneath the IX layers of Rexed laminae. We used the nonparametric one-way ANOVA (Kruskal-Wallis) with a Dunn's post test for multiple comparisons between sham animals and SCI and/or SCI-pharmacologically treated rats.

Motoneuron Culture and Immunocytochemistry. Hb9::GFP transgenic mice were maintained as hemizygotes by crossing with nontransgenic female mice. Mice were housed in a conventional dark/light cycle (dark 1100 hours to 0900 hours) and positive vaginal plugs at 0900 hours were recorded as embryonic day (E)0. Motoneurons were isolated from E12 spinal cords essentially as described previously (6,7). Motoneurons were seeded at a density of about 5.000 cells per well in four-well plates previously treated with polyornithine (Life Technologies) and laminin (Becton Dickinson). Motoneurons were cultured in Neurobasal medium supplemented with 2% (vol/vol) horse serum, 25 μM L-glutamate, 25 μM β-mercaptoethanol, 0.5 mM L-glutamine, 2% (vol/vol) B-27 (all from Life Technologies), and the neurotrophic factors BDNF (1 ng/mL; R&D Systems), ciliary neurotrophic factor (CNTF) (10 ng/mL; R&D Systems), and glial cell line-derived neurotrophic factor (GDNF) (100 pg/mL; Sigma). Half of the culture medium was replaced every 2 d.

At 13 or 14 DIV, cultures were incubated with TCB-2 or mock for 30 min at 37 °C in an incubator containing 5% CO 2 in a humidified air atmosphere. Cultures were fixed for 15 min by adding an equal volume of 7.4% (vol/vol) formaldehyde in PBS, washed and blocked in buffer containing PBS, 0.1% Triton X-100, 1% BSA, and 5% goat serum. Coverslips were incubated overnight at 4 °C with rabbit polyclonal anti-KCC2 antibodies (Millipore) at 1:300 in blocking buffer, washed with PBS, and incubated for 1 h with Cy3-conjugated anti-rabbit antibodies (Jackson Immunoresearch) diluted in blocking buffer. Coverslips were mounted in Vectashield/DAPI solution (Vector Labs) and examined with a confocal fluorescence microscope (60× oil objective; Fluoview; Olympus). Thirty-four randomly chosen cells per condition from two independent experimental series were imaged at 3× magnification using identical settings for laser power, photomultipliers, electronic gain, and offset. For each cell, plasma membrane and cytosol were manually delimited and fluorescence intensities in both areas were quantified using the Neuron J plugin of the ImageJ software (National Institutes of Health). The individuals performing data analysis were blind to the conditions of motoneurons.

In Vitro Electrophysiological Recordings. The aCSF used for perfusion was composed of 130 mM NaCl, 4 mM KCl, 3.75 mM CaCl 2 , 1.3 mM MgSO 4 , 0.58 mM NaH 2 PO 4 , 25 mM NaHCO 3 , and 10 mM glucose and was oxygenated with 95% O 2 and 5% CO 2 (pH 7.4; temperature 24-25 °C). Intracellular recordings. We measured the effects of various pharmacological compounds on the reversal potentials of IPSPs (3) (E IPSP ) from the spinal cord of intact and cord-transected rats (P5-P7). We also measured E IPSP 4-6 d after transection or sham surgery performed at P0. After removing the pia, we recorded lumbar motoneurons intracellularly using glass microelectrodes filled with 2 M K-acetate (90-to 150-MΩ resistance). We recorded intracellular potentials in the discontinuous current-clamp (DCC) mode (Axoclamp 2B amplifier; Digidata 1200 interface; pClamp9 software; Axon Instruments). We considered only neurons exhibiting a stable (>15 min) resting membrane potential for analysis. We identified motoneurons in the L4-L5 segments by their antidromic response to ventral root stimulation. We used glass suction electrodes to stimulate the ipsilateral ventral funiculus at the L2-L3 level. Such stimulations induced GABA A R-and GlyR-mediated IPSPs (3,8,9) in the presence of 2-amino-5-phosphonovaleric acid (AP5) (50-100 μM) and 6-cyano-7-nitroquin-oxaline-2,3-dione (CNQX) (10 μM). We recorded IPSPs at various holding potentials (500-ms-long current pulses) and collected at least 20 values for each motoneuron. We measured and plotted ampli-tudes of IPSPs against holding potentials and obtained E IPSP from the regression line (Prism Version 5; Graphpad). Quantification of polysynaptic responses. We placed monopolar stainless steel electrodes in contact with the spinal roots and insulated them with Vaseline for stimulation and recording (P5-P7). We recorded L4-L5 ventral roots discharges by means of an ACcoupled amplifier (bandwidth: 70 Hz to 3 kHz) in response to the stimulation of the homonymous dorsal root (0.3 ms). Stimulations were delivered at supramaximal intensity every 60 s to avoid the synaptic depression described in this preparation. Once the signal was stable for at least 20 min, we added the pharmacological compound to be tested to the perfusion liquid for 20-30 min. We rectified signals and measured the areas under the curve from 3 to 100 ms after the onset of the monosynaptic signal (pClamp9 software). The effect of the drug was considered 20 min following bath application of the compound. Measurement of RDD in vitro. We delivered supramaximal electrical stimulations (0.3-ms duration) to dorsal roots. We measured the amplitude of the monosynaptic response between the first positive and negative peaks. We delivered 15 stimulations at various frequencies (30 s between each series). We normalized the amplitude of responses (the first three discarded) to the controls evoked at 30-s intervals (mean of five pulses). We took into account at least three series of stimulations before and 20-30 min after the application of the compound. Fictive locomotion. The coordination between opposite L3 ventral root bursts was investigated during fictive locomotion in six transected animals (4) (P5, n = 3; P6, n = 3). Fictive locomotion was elicited by bath application of N-methyl-D, L-aspartate (NMA) (12-16 μM). Analyses consisted in rectifying and integrating the recordings (time constant of 25 ms). A threshold function was used to determine bursts onset and termination. The threshold was usually set to ∼30% of the peak value. The duration of motor bursts was measured, and the middle of bursts was considered to calculate the period (defined as the time between the midpoint of two consecutive bursts) and the phase relationships between the left and right bursts (defined as the time between the midpoint of a burst and that of the next burst in the contralateral root, divided by the period of the ongoing cycle). The coordination was evaluated by means of cross-correlation analysis (pClamp9 software; Axon Instruments). Phase data were multiplied by 360°to be analyzed by means of circular statistics (Oriana; Kovach Computing Services).

In Vivo Electrophysiological Recordings (H Reflex). We repeated a series of measurements throughout the experiment. First, we stimulated the tibial nerve for 0.2 ms at 0.2 Hz with increasing current intensities (until M max was stabilized) and determined the intensity necessary to get a maximal H response. Then, we used this intensity for trains of 20 stimulations at 0.1, 0.5, 1, 2, and 5 Hz to measure the RDD, with at least 1-min intervals between each train of stimulations so that a whole series of measurements lasted about 15min. We added one third of the initial dose of anesthesia every 30 min and maintained the rat temperature around 37 °C. To determine the level of RDD at the different frequencies, we discarded responses to the first three stimulations necessary for the depression to occur and expressed all of the responses as percentages relative to the mean response at 0.2 Hz in the same series of measurements.

We injected the drug or its vehicle after two stable series of measurements and carried on measuring current-voltage (I/V) curves and RDD for 3 h.

Drugs. We purchased DOI hydrochloride, ketanserin tartrate, VU0240551, PDBu, α-methyl 5-HT, NMA, strychnine, and picrotoxin from Sigma. We purchased TCB-2, MDL11,939, SB206553 hydrochloride, chelerythrine chloride, Gö6976, and FR236924 (DCP-LA) from Tocris. DL-AP5 and CNQX were purchased from Abcam. All compounds were prepared in aCSF except VU0240551, PDBu, α-methyl 5-HT, MDL11,939, SB206553 hydrochloride, chelerythrine chloride, Gö6976, and FR236924, which were diluted in DMSO (0.1% final concentration).

Statistical Analyses. Group measurements are expressed as means ± SEM. We used t tests to compare two groups and one-or two-way ANOVA to compare more than two groups, or corresponding nonparametric analyses when data were not nor-mally distributed or when small samples (<20) were analyzed (Prism 5 software; Graphpad). Nonlinear regressions were performed to compare the levels of H-reflex depression at all stimulation frequencies tested in different groups. A logarithmic transformation of frequency values was also performed to enable linear regression analyses and comparison of slopes. Statistical tests used in each case are given in figure legends. The level of significance was set at P < 0.05. Fig. S1. TCB-2 has no effect on several electrical properties of motoneurons. (A) V rest of six motoneurons (control rats; P5-P7) before and 20-30 min after starting bath application of TCB-2 (0.1 μM diluted in aCSF). (B and C) TCB-2 has a significant effect neither on the rheobase (defined as the minimum current required to induce action potential during a 500 ms pulse; n = 5) (B) nor on the firing rate (n = 5) (C). The firing rate was recorded at 1.5-twofold the rheobase in the both conditions. ns, P > 0.05 (Wilcoxon paired test). ) on E IPSP in motoneurons recorded from control rats (P5-P7; n = 8). E IPSP was significantly more depolarized after application of the 5-HT 2B/2C R agonist α-methyl 5-HT (0.5-5 μM) compared with control (P5-P6 rats; n = 6). These results suggest that 5-HT 2B R and/or 5-HT 2C R exert a depolarizing action on E IPSP . ns, P > 0.05; *P < 0.05; **P < 0.01 (Wilcoxon paired test). Fig. S3. TCB-2 has no effect on IPSP kinetics. (A) IPSP recorded by means of a microelectrode filled with KCl (2 M) to load the cell with Cl -and, thereby, shift E IPSP . The IPSP was quite similar before and after adding TCB-2 (0.1 μM). No significant difference was observed on both the amplitude (n = 6) (Center) and the duration (n = 6) (Right) of IPSPs from newborn rats (P4-P5). (B) The area of IPSPs was measured at V rest before and after adding TCB-2 in the continuous presence of VU0240551 at 25 μM (P5-P6 rats; n = 11). ns, P > 0.05 (Wilcoxon paired test). Fig. S4. RDD of the monosynaptic reflex in vitro is reduced when KCC2 function is genetically or pharmacologically reduced. (A) To investigate the role of KCC2 in this property of spinal cord reflexes, we used Slc12a5 +/-mice (n = 7) and Slc12a5 -/-mice (1) (n = 4), which retain ∼50% and 10% of normal KCC2 protein amounts in the spinal cord, respectively (2). RDD was significantly smaller in both types of mice compared with wild-type mice (n = 9), confirming that KCC2 cotransporters play a key role in the depression of the response. (B) The specific blockage of KCC2 by VU0240551 (25 μM; P5-P6 rats; n = 4) significantly decreases the RDD of the monosynaptic reflex. ns, P > 0.05; *P < 0.05; ***P < 0.001 (one-phase exponential decay regression).

Fig. 1 .

 1 Fig. 1. Activation of 5-HT 2 Rs hyperpolarizes E IPSP and increases membrane expression of KCC2. (A) IPSPs evoked by stimulation of the ventral funiculus of the spinal cord (arrow) at different holding potentials in a motoneuron from a P6 intact rat before and after adding DOI (10 μM). (B) Time course of the change in E IPSP and V rest . (C) E IPSP and driving force (E IPSP -V rest ) measured in control conditions (n = 21) and after adding DOI (n = 8). ***P < 0.001 (Mann-Whitney test). Six motoneurons were tested before and after DOI. *P < 0.05 (Wilcoxon paired test). The effects of DOI were prevented in the presence of ketanserin (10 μM; n = 4) (Center). (D) Effects of DOI (1-1.5 μM) on E IPSP and driving force in motoneurons recorded 4-6 d after neonatal SCI (17 and 6 cells recorded in the absence and the presence of DOI, respectively). **P < 0.01, *P < 0.05 (Mann-Whitney test); *P < 0.05 (Wilcoxon paired test). (E) E IPSP was significantly more hyperpolarized after chronic treatment of DOI (n = 9) compared with SCI animals (n = 22) but not different compared with control animals (n = 21). ns, not significant (P > 0.05); ***P < 0.001 (one-way ANOVA, Tukey's post tests). (F, Upper) Western blots of membrane and cytoplasmic fractions of lumbar spinal cords labeled with a KCC2-specific antibody. The 130-to 140-kDa and the >200-kDa bands correspond to the monomeric and oligomeric proteins, respectively (53). (F, Lower) Quantification of KCC2 expression in DOI-treated rats after neonatal SCI (percentage of untreated rats). *P < 0.05 (Mann-Whitney test; n = 6 in each group). (G) Dual labeling of GlyRα1 and KCC2 on FB-labeled ankle extensor motoneurons, in three conditions (P7): intact, neonatal SCI, and chronic DOI treatment. (Scale bars: 10 μm.) (H) Quantification of the density of membrane KCC2 labeling (ratios of labeled pixel surface per somatic perimeter) in intact rats (n = 34 motoneurons) and untreated (n = 41) or DOI-treated (n = 44) rats with neonatal SCI (three rats in each group). ***P < 0.001 (Kruskal-Wallis test, Dunn's post tests). (I) Ventral part of the lumbar spinal cord exhibiting KCC2-immunopositive dendrites stretching out through the white matter. (Scale bars: 100 μm.) (J) Quantification of the KCC2 labeling in the white matter in intact rats (n = 34) and untreated (n = 41) or DOI-treated (n = 44) rats with neonatal SCI. ***P < 0.001 (Kruskal-Wallis test, Dunn's post tests).

Fig. 2 .

 2 Fig. 2. Involvement of 5-HT 2A Rs via a PKC-dependent signaling pathway. (A) TCB-2 (0.1 μM; 30 min) induces a hyperpolarizing shift of E IPSP without concomitant effect on V rest (P6). (B, Left) Effect of TCB-2 (0.1 μM, gray; 10 μM, black) on 10 motoneurons recorded from control rats (P5-P6). ***P < 0.001 (Mann-Whitney test; n = 12 and n = 14 before and under TCB-2, respectively); **P < 0.01 (Wilcoxon paired test). The difference was also significant when considering only the effect of the lowest concentration [0.1 μM; n = 6; *P < 0.05 (Wilcoxon paired test)]. (B, Right) Effect of TCB-2 (0.1-10 μM) on the driving force (control, n = 12; TCB-2, n = 14). *P < 0.05 (Mann-Whitney test); **P < 0.01 (Wilcoxon paired test). (C) Effect of TCB-2 (0.1 μM) on E IPSP and the driving force of motoneurons (six and nine in the absence and in the presence of TCB-2, respectively) in animals with SCI at birth. ***P < 0.001, *P < 0.05 (Mann-Whitney test); *P < 0.05 (Wilcoxon paired test) (n = 6). (D) E IPSP was significantly depolarized by the application of the KCC2 blocker VU0240551 (25 μM), which prevented the hyperpolarizing effect of TCB-2 (0.1 μM; n = 11). Averages are taken from 3 motoneurons in control situation, 9 motoneurons under VU0240551, and 11 motoneurons in the presence of both VU0240551 and TCB-2. ns, P > 0.05; **P < 0.01 (Mann-Whitney test). (E) TCB-2 (0.1 μM; 30 min) significantly increased the expression of KCC2 at the plasma membrane of Hb9::eGFP transgenic motoneurons in culture (14 DIV). (E, Left and Center) Merge images (Insets) show the KCC2 immunolabeling at the periphery of GFP-expressing cytosol. (E, Right) Ratio of KCC2 expression between plasma membrane and cytosol. ***P < 0.001 (t test; n = 34 cells from two experiments analyzed in each condition). (F) Preincubation of the PKC inhibitor chelerythrine (20 μM; 30 min; n = 6) prevented the effect of DOI (10 μM; n = 3; gray) or TCB-2 (0.1 μM; n = 3). PDBu significantly hyperpolarized E IPSP . ns, P > 0.05; *P < 0.05 (Wilcoxon paired test). The Ca 2+ -dependent PKC inhibitor Gö6976 (2 μM; n = 8) induced a slight but significant hyperpolarization of E IPSP and a further ∼4-mV negative shift was observed after adding TCB-2 (0.1 μM; n = 16). **P < 0.001 (Mann-Whitney test); *P < 0.05 (Wilcoxon paired test). The activator of the Ca 2+ -independent PKCe (FR236924; 2-8 μM; n = 7) hyperpolarized E IPSP. *P < 0.05 (Wilcoxon paired test).

Fig. 3 .

 3 Fig.3. Effects of TCB-2 on ventral root responses to dorsal root stimulation in the intact spinal cord in vitro. (A) Responses evoked in the L5 ventral root by supramaximal stimulation of the ipsilateral homonymous dorsal root, in the in vitro spinal cord isolated from rats at P6. The earliest component represents the monosynaptic response of motoneurons. The amplitude of this response decreased when the dorsal root was stimulated repeatedly (response to the 15th pulse at 1 Hz is shown in blue, superimposed with the control response to the first pulse). The slight increase in latency is attributable to a delayed firing of motoneurons as the pulse number increases. The amplitude of the response to the 15th pulse was much smaller after adding TCB-2 (0.1-0.15 μM). (B) Relative amplitudes of the monosynaptic responses delivered every 30 s before and 20-30 min after adding TCB-2 [percentage of control before TCB-2; P > 0.05 (Wilcoxon paired test); n = 12]. (C) Relative amplitudes of the monosynaptic responses to 15 consecutive stimulations at different frequencies (0.1 Hz, black; 1 Hz, red; 5 Hz, blue) before (continuous line) and after adding TCB-2 (dotted line). This is the same experiment as in A. (D) Mean (± SEM) relative amplitudes of the monosynaptic reflex at different stimulation frequencies in seven animals at P5-P6 before [controls in artificial cerebrospinal fluid (aCSF)] and after TCB-2 application (0.1-0.15 μM). Twelve values (the first three discarded) were averaged for each animal. The RDD was significantly increased by TCB-2. ***P < 0.001 (onephase exponential decay regression). Note that for each frequency of stimulation, except at 0.1 Hz, the difference between before and after TCB-2 was significant [P < 0.05 (Wilcoxon paired test)]. (E) Mean envelopes of the ventral root (VR) burst evoked by stimulation of the ipsilateral homonymous dorsal root (DR) before and after adding TCB-2 (0.15 μM). *P < 0.05 (Wilcoxon paired test; n = 7).

Fig. 4 .

 4 Fig. 4. Activation of 5-HT 2A Rs increases the RDD of the H reflex. (A) H max responses evoked in adult rats after SCI before (Left) and 7 min after i.p. injection of TCB-2 (0.3 mg/kg) (Right). Each trace is the mean response to 17 consecutive stimulations (the first three discarded) at 0.1, 0.5, 1, 2, or 5 Hz. (B) Time course of the change in H-reflex depression after i.p. injection of vehicle (NaCl; n = 6 rats) or TCB-2 (n = 4). (C) Mean relative amplitudes of the H reflex before (black) and after (blue, 7 min; red, 22 min) the injection of vehicle (Left) or TCB-2 (Right).The data of all experiments are represented (the dots that are slightly offset at each frequency of stimulation are for clarity) together with the one-phase exponential decay fit of these data. ***P < 0.001, comparison of the fits before and after TCB-2 (either 7 or 22 min). The fits before and after vehicle are not significantly different.

Fig. S2 .

 S2 Fig. S2. Relative effects of blocking or activating 5-HT 2A R and 5-HT 2B/C R on E IPSP. (A) Preincubation during 30 min of the 5-HT 2 R antagonist ketanserin (1 μM; n = 4) or the more specific 5-HT 2A R antagonist MDL11,939 (2 μM; n = 4) prevented the hyperpolarizing effect of TCB-2 (0.1 μM) on E IPSP . The latter compound had no effect on E IPSP (-70.6 and -70.8 mV in control and under MDL11,939, respectively; n = 4). TCB-2 (0.1 μM) still induced a hyperpolarizing shift of E IPSP in the presence of the 5-HT 2B-C R antagonist SB206553 (3-5 μM; n = 6). (B) Hyperpolarizing effect of the 5-HT 2B/2C R antagonist SB206553 (3-5 μM) on E IPSP in motoneurons recorded from control rats (P5-P7; n = 8). E IPSP was significantly more depolarized after application of the 5-HT 2B/2C R agonist α-methyl 5-HT (0.5-5 μM) compared with control (P5-P6 rats; n = 6). These results suggest that 5-HT 2B R and/or 5-HT 2C R exert a depolarizing action on E IPSP . ns, P > 0.05; *P < 0.05; **P < 0.01 (Wilcoxon paired test).

1 .

 1 Stil A, et al. (2011) Contribution of the potassium-chloride co-transporter KCC2 to the modulation of lumbar spinal networks in mice. Eur J Neurosci 33(7):1212-1222. 2. Woo NS, et al. (2002) Hyperexcitability and epilepsy associated with disruption of the mouse neuronal-specific K-Cl cotransporter gene. Hippocampus 12(2):258-268.

Fig. S5 .

 S5 Fig.S5. Blocking KCC2 prevents the effect of TCB-2 on the RDD of the monosynaptic reflex in vitro. TCB-2 (0.1 μM) did not increase the RDD of the monosynaptic response in the continuous presence of the specific blocker of KCC2 (VU0240551; 25 μM; P5-P6 rats; n = 4). ns, P > 0.05 (Wilcoxon paired test).

Fig. S6 .

 S6 Fig. S6. TCB-2 reduces polysynaptic ventral root responses through a modulation of chloride homeostasis. (A) The specific blocker of KCC2 (VU0240551; 25 μM) significantly increased the polysynaptic ventral root responses and prevented the effect of TCB-2 on the polysynaptic response (P5 rats). ns, P > 0.05; **P < 0.01 (Kruskal-Wallis test with Dunn's post test; n = 4 in each group). (B) The polysynaptic ventral root responses (rectified averaged signals) was significantly increased in presence of the GlyR and GABA receptor antagonists [strychnine and picrotoxin (PTX), respectively] compared with control conditions (P5-P6 rats). The decrease of the polysynaptic response by TCB-2 (0.1 μM) (Fig. 4) was prevented in the continuous presence of strychnine and PTX. ns, P > 0.05; *P < 0.05 (Kruskal-Wallis test with Dunn's post test; n = 4 in each group).

Fig. S7 .

 S7 Fig. S7. Activation of 5-HT 2A Rs in vitro restores the left-right alternating locomotor pattern after neonatal spinal cord transection. (A) Integrated recordings from the left and right L3 ventral roots (VR) at P5 in the presence of NMA alone (16 μM) (Left) or together with 5-HT 2A R agonist TCB-2 (0.1 μM) (Center). Gray areas indicate the bursts occurring in the left L3 VR. Cross-correlograms between the two VR signals computed from 10 min of activity induced by NMA alone (black line) or NMA and TCB-2 (gray line). (B) Time course of the changes of cross-correlation coefficient in six transected rats in NMA alone (10-16 μM, black plot) or NMA and TCB-2 (0.1 μM, gray plot). Mean cross-correlation coefficient (Right) between left and right ventral root activities computed when activities were stable in both conditions (5-10 min). *P < 0.05 (Wilcoxon paired test; n = 6). (C) Distribution of phase relationships between left and right ventral root bursts in NMA alone (Left) and NMA plus TCB-2 (Right) in all of the spinal animals (P5-P6; n = 6).

| www.pnas.org/cgi/doi/10.1073/pnas.1213680110 Bos et al.

| www.pnas.org/cgi/doi/10.1073/pnas.1213680110 Bos et al.

Bos et al. www.pnas.org/cgi/content/short/1213680110

ACKNOWLEDGMENTS. We thank Mouloud Bouhadfane for helpful discussions throughout the experiments and Florian Gackière and Frédéric Brocard for their comments on the manuscript. This work was supported by the Christopher and Dana Reeve Foundation Grants VB1-0502-2 and VB2-0801-2, the International Spinal Research Trust Grant STR110, the French Agence Nationale pour la Recherche Grant ANR-2010-BLAN-1407-01, and the French Institut pour la Recherche sur la Moelle épinière et l'Encéphale (to L.V.). P.B., G.H., and H.B. are supported by the Institut National de la Santé et de la Recherche Médicale, and K.S. is supported by Association Française contre les Myopathies Grant 13912.

Supporting Information

The TCB-2 injection was 0.3 mg/kg, i.p. *None of the paired comparison was significant (P > 0.05; n = 4).